Derivatives of dolaproine-dolaisoleucine peptides

ABSTRACT

Provided are peptide analogs, pharmaceutical compositions comprising such compounds, and methods of treating cancer with such compounds.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.15/313,906, which is a U.S. National Stage of International PatentApplication No. PCT/US2015/032704, filed May 27, 2015, which claims thebenefit of U.S. Provisional Patent Application No. 62/004,084, filed May28, 2014, the disclosure of each of which is incorporated by referenceherein in its entirety.

FIELD OF INVENTION

Provided herein are dolaproine-dolaisoleuine peptide analogs,pharmaceutical compositions comprising such compounds, and methods oftreating cancer with such compounds.

BACKGROUND

Cancer is the second leading cause of human death exceeded only bycoronary disease. In the U.S., cancer accounts for nearly 1 in 4 deaths.Worldwide, millions of people die from cancer every year. In the UnitedStates alone, as reported by the American Cancer Society, cancer causesthe death of well over a half-million people annually, with over 1.5million new cases diagnosed per year. While deaths from heart diseasehave been declining significantly, those resulting from cancer generallyare on the rise. In the early part of the next century, cancer ispredicted to become the leading cause of death unless new medicines arefound.

Worldwide, several cancers stand out as the leading killers. Inparticular, carcinomas of the lung, prostate, breast, colon, pancreas,ovary, and bladder represent the primary causes of cancer death. Withvery few exceptions, metastatic cancer is fatal. Moreover, even forthose cancer patients who initially survive their primary cancers,common experience has shown that their lives are dramatically altered.Many cancer patients experience strong anxieties driven by the awarenessof the potential for recurrence or treatment failure. Many cancerpatients experience physical debilitations following treatment.Furthermore, many cancer patients experience a recurrence.

Promising new cancer therapeutics include the dolastatins and syntheticdolastatin analogs such as auristatins (U.S. Pat. Nos. 5,635,483,5,780,588, 6,323,315, and 6,884,869; Shnyder et al. (2007) Int. J.Oncol. 31:353-360; Otani, M. et al. Jpn. J. Cancer Res. 2000, 91,837-844; PCT Intl. Publ. Nos. WO 01/18032 A3, WO 2005/039492, WO2006/132670, and WO 2009/095447; Fennell, B. J. et al. J. Antimicrob.Chemther. 2003, 51, 833-841). Dolastatins and auristatins have beenshown to interfere with microtubule dynamics, thus disrupting celldivision (Woyke et al. (2001) Antimicrob. Agents Chemother.45(12):3580-3584), and have anticancer (U.S. Pat. No. 5,663,149) andantifungal activity (Pettit et al. (1998) Antimicrob. Agents Chemother.42:2961-2965). Unfortunately, despite early enthusiasm, dolastatin 10showed poor results as a single agent in phase II clinical trials(Shnyder (2007), supra). Certain compounds in the auristatins familyhave shown greater promise as clinical candidates with improved efficacyand pharmacological characteristics over the dolastatins (Pettit et al.(1995) Anti-Cancer Drug Des. 10:529-544; Pettit et al. (1998)Anti-Cancer Drug Des. 13:243-277; Shnyder (2007), supra). Varioussynthetic analogs of this structural type have been described (U.S. Pat.Nos. 6,569,834; 6,124,431; and Pettit et al. (2011) J. Nat. Prod.74:962-968).

The auristatins have several properties which make them attractive forpharmaceutical development. First, these compounds are extremely potent.Second, their preparation is straight-forward because of the peptidicscaffold. Third, they possess good pharmacokinetic and metabolicprofiles compared to peptides in general, or to other cancer drugclasses in particular.

Despite significant advances, there remains a need for new anticancertherapeutics with desirable pharmaceutical properties.

SUMMARY

Provided herein are novel dolaproine-dolaisoleuine peptide analogs.Thus, provided herein are compounds of Formula (I):

whereinR¹ and R² are each independently —H or alkyl;X is —O—, —NR^(z)—, —S—, or is absent;

wherein R^(z) is —H or alkyl;

R³ is a group of the formula:

wherein R¹⁵ and R¹⁶ are each independently —H, —OH, —NH₂, —SH, —N₃,alkyl, alkenyl, alkynyl, -alkyl-OH, -alkyl-NH₂, -alkyl-SH, or -alkyl-N₃;R⁴ is a group of the formula:

wherein R¹⁷ and R¹⁸ are each independently —H, —OH, —NH₂, —SH, —N₃,—CO₂H, alkyl, alkenyl, alkynyl, -alkyl-OH, -alkyl-NH₂, -alkyl-SH,-alkyl-N₃ or -alkyl-CO₂HR⁵ is sec-butyl or isobutyl;R⁶ is —H or alkyl;R⁷ and R⁸ are each independently —H, alkyl, —CO₂R^(a), CONR^(b)R^(c),substituted or unsubstituted phenyl, or substituted or unsubstitutedheterocyclic ring;wherein R^(a) is —H or alkyl;R^(b) and R^(c) are each independently H or alkyl;R⁹ is —H or alkyl; or R⁹ is taken together with R⁴ and the atoms towhich they are attached to form a substituted or unsubstitutedheterocycloalkyl ring;R¹⁰ is —H or alkyl;R¹¹ is —H or alkyl;R¹² is —H or alkyl;R¹³ is —H or alkyl; andR¹⁴ is —H, —OH or alkyl;provided that when X is absent and R¹⁵, R¹⁶, R¹⁷ and R¹⁸ are eachmethyl, then R⁸ is not substituted or unsubstituted phenyl, orsubstituted or unsubstituted heterocyclic ring; or a pharmaceuticallyacceptable salt thereof.

Also provided herein is a pharmaceutical composition comprising aneffective amount of at least one compound of Formula (I), or apharmaceutically acceptable salt thereof, and a pharmaceuticallyacceptable excipient.

Also provided herein is a method of treating a subject suffering from ordiagnosed with cancer, comprising administering to a subject in need ofsuch treatment an effective amount of at least one compound of Formula(I), or a pharmaceutically acceptable salt thereof.

Also provided herein is use of at least one compound of Formula (I), ora pharmaceutically acceptable salt thereof, for treatment of cancer in asubject in need of such treatment.

Also provided herein is use of at least one compound of Formula (I), ora pharmaceutically acceptable salt thereof, in the manufacture of amedicament for treatment of cancer in a subject in need of suchtreatment.

Also provided herein is a kit containing at least one compound ofFormula (I), or a pharmaceutically acceptable salt thereof, for use intreating cancer in a subject in need of such treatment, and instructionsfor use.

Also provided herein is an article of manufacture comprising at leastone compound of Formula (I), or a pharmaceutically acceptable saltthereof, for use in treating cancer in a subject in need of suchtreatment.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows in vitro tubulin polymerization data for tubulin treatedwith Example 2, Example 4, and Example 6. Untreated (buffer) tubulinshows the basal level of tubulin polymerization. A tubulin stabilizer(Paclitaxel) and a tubulin de-stabilizer (Control) were used ascontrols. All compounds were used at a final concentration of 10 μM.

FIG. 2 shows in vitro tubulin polymerization data for tubulin treatedwith Example 1, Example 3, and Example 5. Untreated (buffer) tubulinshows the basal level of tubulin polymerization. A tubulin stabilizer(Paclitaxel) and a tubulin de-stabilizer (Control) were used ascontrols. All compounds were used at a final concentration of 10 μM.

FIG. 3 shows in vitro tubulin polymerization data for tubulin treatedwith Example 8, Example 9, and Example 12. Untreated (buffer) tubulinshows the basal level of tubulin polymerization. A tubulin stabilizer(Paclitaxel) and a tubulin de-stabilizer (Control) were used ascontrols. All compounds were used at a final concentration of 10 μM.

FIG. 4 shows in vitro tubulin polymerization data for tubulin treatedwith Example 7, Example 10, and Example 11. Untreated (Buffer) tubulinshows the basal level of tubulin polymerization. A tubulin stabilizer(Paclitaxel) and a tubulin de-stabilizer (Control) were used ascontrols. All compounds were used at a final concentration of 10 μM.

FIG. 5 shows in vitro tubulin polymerization data for tubulin treatedwith Example 19, Example 20, and Example 21. Untreated (Buffer) tubulinshows the basal level of tubulin polymerization. A tubulin stabilizer(Paclitaxel) and a tubulin de-stabilizer (Control) were used ascontrols. All compounds were used at a final concentration of 10 μM.

FIG. 6 shows in vitro tubulin polymerization data for tubulin treatedwith Example 13, Example 16, and Example 22. Untreated (Buffer) tubulinshows the basal level of tubulin polymerization. A tubulin stabilizer(Paclitaxel) and a tubulin de-stabilizer (Control) were used ascontrols. All compounds were used at a final concentration of 10 μM.

FIG. 7 shows in vitro tubulin polymerization data for tubulin treatedwith Example 14, Example 15, Example 18, Example 20, and Example 23.Untreated (Buffer) tubulin shows the basal level of tubulinpolymerization. A tubulin stabilizer (Paclitaxel) and a tubulinde-stabilizer (Control) were used as controls. All compounds were usedat a final concentration of 10 μM.

FIG. 8 shows in vitro tubulin polymerization data for tubulin treatedwith Example 17, Example 19, Example 25, and Example 26. Untreated(Buffer) tubulin shows the basal level of tubulin polymerization. Atubulin stabilizer (Paclitaxel) and a tubulin de-stabilizer (Control)were used as controls. All compounds were used at a final concentrationof 10 μM.

FIG. 9 shows the results for Examples 1, 2, 3 and Paclitaxel in an invitro cytotoxicity experiment using PC3 cells, as described in ExampleB1. Data is graphed as percent survival versus concentration of testcompound, compared to untreated control wells.

FIG. 10 shows the results for Examples 1, 2, 3 and Paclitaxel in an invitro cytotoxicity experiment using HCT15 cells, as described in ExampleB1. Data is graphed as percent survival versus concentration of testcompound, compared to untreated control wells.

FIG. 11 shows the results for Examples 1, 2, 3 and Paclitaxel in an invitro cytotoxicity experiment using HCC-1954 cells, as described inExample B1. Data is graphed as percent survival versus concentration oftest compound, compared to untreated control wells.

FIG. 12 shows the results for Examples 4, 5, and 6 in an in vitrocytotoxicity experiment using PC3 cells, as described in Example B1.Data is graphed as percent survival versus concentration of testcompound, compared to untreated control wells.

FIG. 13 shows the results for Examples 4, 5, and 6 in an in vitrocytotoxicity experiment using HCT15 cells, as described in Example B1.Data is graphed as percent survival versus concentration of testcompound, compared to untreated control wells.

FIG. 14 shows the results for Examples 4, 5, and 6 in an in vitrocytotoxicity experiment using HCC-1954 cells, as described in ExampleB1. Data is graphed as percent survival versus concentration of testcompound, compared to untreated control wells.

FIG. 15 shows the results for Examples 7, 8, and 9 in an in vitrocytotoxicity experiment using PC3 cells, as described in Example B1.Data is graphed as percent survival versus concentration of testcompound, compared to untreated control wells.

FIG. 16 shows the results for Examples 7, 8, and 9 in an in vitrocytotoxicity experiment using HCT15 cells, as described in Example B1.Data is graphed as percent survival versus concentration of testcompound, compared to untreated control wells.

FIG. 17 shows the results for Examples 7, 8, and 9 in an in vitrocytotoxicity experiment using HCC-1954 cells, as described in ExampleB1. Data is graphed as percent survival versus concentration of testcompound, compared to untreated control wells.

FIG. 18 shows the results for Examples 10, 11, and 12 in an in vitrocytotoxicity experiment using PC3 cells, as described in Example B1.Data is graphed as percent survival versus concentration of testcompound, compared to untreated control wells.

FIG. 19 shows the results for Examples 10, 11, and 12 in an in vitrocytotoxicity experiment using HCT15 cells, as described in Example B1.Data is graphed as percent survival versus concentration of testcompound, compared to untreated control wells.

FIG. 20 shows the results for Examples 10, 11, and 12 in an in vitrocytotoxicity experiment using HCC-1954 cells, as described in ExampleB1. Data is graphed as percent survival versus concentration of testcompound, compared to untreated control wells.

FIG. 21 shows the results for Examples 13, 14, and 15 in an in vitrocytotoxicity experiment using PC3 cells, as described in Example B1.Data is graphed as percent survival versus concentration of testcompound, compared to untreated control wells.

FIG. 22 shows the results for Examples 13, 14, and 15 in an in vitrocytotoxicity experiment using HCC-1954 cells, as described in ExampleB1. Data is graphed as percent survival versus concentration of testcompound, compared to untreated control wells.

FIG. 23 shows the results for Examples 16, 21, and 22 in an in vitrocytotoxicity experiment using PC3 cells, as described in Example B1.Data is graphed as percent survival versus concentration of testcompound, compared to untreated control wells.

FIG. 24 shows the results for Examples 16, 21, and 22 in an in vitrocytotoxicity experiment using HCC-1954 cells, as described in ExampleB1. Data is graphed as percent survival versus concentration of testcompound, compared to untreated control wells.

FIG. 25 shows the results for Examples 17, 18, and 19 in an in vitrocytotoxicity experiment using PC3 cells, as described in Example B1.Data is graphed as percent survival versus concentration of testcompound, compared to untreated control wells.

FIG. 26 shows the results for Examples 17, 18, and 19 in an in vitrocytotoxicity experiment using HCC-1954 cells, as described in ExampleB1. Data is graphed as percent survival versus concentration of testcompound, compared to untreated control wells.

FIG. 27 shows the results for Examples 20 and 23 in an in vitrocytotoxicity experiment using PC3 cells, as described in Example B1.Data is graphed as percent survival versus concentration of testcompound, compared to untreated control wells.

FIG. 28 shows the results for Examples 20 and 23 in an in vitrocytotoxicity experiment using HCC-1954 cells, as described in ExampleB1. Data is graphed as percent survival versus concentration of testcompound, compared to untreated control wells.

FIG. 29 shows the results for Examples 25 and 26 in an in vitrocytotoxicity experiment using PC3 cells, as described in Example B1.Data is graphed as percent survival versus concentration of testcompound, compared to untreated control wells.

FIG. 30 shows the results for Examples 25 and 26 in an in vitrocytotoxicity experiment using HCC-1954 cells, as described in ExampleB1. Data is graphed as percent survival versus concentration of testcompound, compared to untreated control wells.

DETAILED DESCRIPTION

For the sake of brevity, the disclosures of the publications cited inthis specification, including patents, are herein incorporated byreference.

As used herein, the terms “including,” “containing,” and “comprising”are used in their open, non-limiting sense.

To provide a more concise description, some of the quantitativeexpressions given herein are not qualified with the term “about”. It isunderstood that, whether the term “about” is used explicitly or not,every quantity given herein is meant to refer to the actual given value,and it is also meant to refer to the approximation to such given valuethat would reasonably be inferred based on the ordinary skill in theart, including equivalents and approximations due to the experimentaland/or measurement conditions for such given value.

The term “alkyl,” by itself or as part of another term, refers to asaturated C₁-C₁₂ hydrocarbon containing normal, secondary, tertiary orcyclic carbon atoms. Particular alkyl groups are those having 1 to 8carbon atoms, 1 to 6 carbon atoms, or 1 to 4 carbon atoms.

Examples of alkyl groups include, but are not limited to: methyl (Me),ethyl (Et), n-propyl, isopropyl, butyl, isobutyl, sec-butyl, tert-butyl(tBu), n-pentyl, isopentyl, tert-pentyl, and n-hexyl, isohexyl. In someembodiments, an alkyl group has normal, secondary, or tertiary carbonatoms and does not have cyclic carbon atoms.

The term “alkenyl,” by itself or as part of another term, refers to aC₂-C₁₂ hydrocarbon containing normal, secondary, tertiary or cycliccarbon atoms with at least one site of unsaturation, i.e., acarbon-carbon, sp² double bond. Particular alkenyl groups are thosehaving 2 to 8 carbon atoms, 2 to 6 carbon atoms, or 2 to 4 carbon atoms.Examples include, but are not limited to: vinyl (—CH═CH₂), allyl(—CH₂CH₂═CH₂), cyclopentenyl (—C₅H₇), and 5-hexenyl(—CH₂CH₂CH₂CH₂CH═CH₂). In some embodiments, an alkenyl group has normal,secondary, or tertiary carbon atoms and does not have cyclic carbonatoms.

The term “alkynyl,” by itself or as part of another term, refers to aC₂-C₁₂ hydrocarbon containing normal, secondary, tertiary or cycliccarbon atoms with at least one site of unsaturation, i.e., acarbon-carbon, sp triple bond. Particular alkynyl groups are thosehaving 2 to 8 carbon atoms, 2 to 6 carbon atoms, or 2 to 4 carbon atoms.Examples include, but are not limited to: ethynyl (—C≡CH) and 2-propynyl(—CH₂C≡CH). In some embodiments, an alkynyl group has normal, secondary,or tertiary carbon atoms and does not have cyclic carbon atoms.

The term “alkoxy” refers to an —O-alkyl group, where the O is the pointof attachment to the rest of the molecule, and alkyl is as definedabove.

The term “heterocycloalkyl” refers to a monocyclic, or fused, bridged,or spiro polycyclic ring structure that is saturated or partiallysaturated and has from 3 to 12 ring atoms per ring structure selectedfrom carbon atoms and up to three heteroatoms selected from nitrogen,oxygen, and sulfur. Particular heterocycloalkyl groups are those havingfrom 3 to 8 ring atoms or from 5 to 7 ring atoms per ring structure. Thering structure may optionally contain up to two oxo groups on carbon orsulfur ring members. Illustrative entities, in the form of properlybonded moieties, include:

The term “heteroaryl” refers to a monocyclic, fused bicyclic, or fusedpolycyclic aromatic heterocycle (ring structure having ring atomsselected from carbon atoms and up to four heteroatoms selected fromnitrogen, oxygen, and sulfur) having from 3 to 12 ring atoms perheterocycle. Particular heteroaryl groups are those having from 3 to 8ring atoms or from 5 to 7 ring atoms per ring structure. Illustrativeexamples of heteroaryl groups include the following entities, in theform of properly bonded moieties:

The terms “heterocycle,” “heterocyclic,” or “heterocyclyl” as usedherein encompass both the “heterocycloalkyl” and “heteroaryl” moietiesas defined above.

Those skilled in the art will recognize that the species ofheterocyclyl, heteroaryl and heterocycloalkyl groups listed orillustrated above are not exhaustive, and that additional species withinthe scope of these defined terms may also be selected.

The term “halogen” represents chlorine, fluorine, bromine, or iodine.The term “halo” represents chloro, fluoro, bromo, or iodo.

The term “substituted” means that the specified group or moiety bearsone or more substituents. The term “unsubstituted” means that thespecified group bears no substituents. The term “optionally substituted”means that the specified group is unsubstituted or substituted by one ormore substituents. Where the term “substituted” is used to describe astructural system, the substitution is meant to occur at anyvalency-allowed position on the system.

Any formula given herein is intended to represent compounds havingstructures depicted by the structural formula as well as certainvariations or forms. In particular, compounds of any formula givenherein may have asymmetric centers and therefore exist in differentenantiomeric forms. All optical isomers and stereoisomers of thecompounds of the general formula, and mixtures thereof, are consideredwithin the scope of the formula. Thus, any formula given herein isintended to represent a racemate, one or more enantiomeric forms, one ormore diastereomeric forms, one or more atropisomeric forms, and mixturesthereof. Furthermore, certain structures may exist as geometric isomers(i.e., cis and trans isomers), as tautomers, or as atropisomers.Additionally, any formula given herein is intended to refer also to anyone of hydrates, solvates, and amorphous and polymorphic forms of suchcompounds, and mixtures thereof, even if such forms are not listedexplicitly. In some embodiments, the solvent is water and the solvatesare hydrates.

Any formula given herein is also intended to represent unlabeled formsas well as isotopically labeled forms of the compounds. Isotopicallylabeled compounds have structures depicted by the formulas given hereinexcept that one or more atoms are replaced by an atom having a selectedatomic mass or mass number. Examples of isotopes that can beincorporated into compounds described herein include isotopes ofhydrogen, carbon, nitrogen, oxygen, phosphorous, fluorine, chlorine, andiodine, such as ²H, ³H, ¹¹C, ¹³C, ¹⁴C, ¹⁵N, ¹⁸O, ¹⁷O, ³¹P, ³²P, ³⁵S,³⁶Cl, and ¹²⁵I, respectively. Such isotopically labeled compounds areuseful in metabolic studies (preferably with ¹C), reaction kineticstudies (with, for example ²H or ³H), detection or imaging techniques[such as positron emission tomography (PET) or single-photon emissioncomputed tomography (SPECT)] including drug or substrate tissuedistribution assays, or in radioactive treatment of patients. Inparticular, an ¹⁸F or ¹¹C labeled compound may be particularly preferredfor PET or SPECT studies. Further, substitution with heavier isotopessuch as deuterium (i.e., ²H) may afford certain therapeutic advantagesresulting from greater metabolic stability, for example increased invivo half-life or reduced dosage requirements. Isotopically labeledcompounds described herein and prodrugs thereof can generally beprepared by carrying out the procedures disclosed in the schemes or inthe examples and preparations described below by substituting a readilyavailable isotopically labeled reagent for a non-isotopically labeledreagent.

When referring to any formula given herein, the selection of aparticular moiety from a list of possible species for a specifiedvariable is not intended to define the same choice of the species forthe variable appearing elsewhere. In other words, where a variableappears more than once, the choice of the species from a specified listis independent of the choice of the species for the same variableelsewhere in the formula, unless stated otherwise.

The nomenclature “C_(i-j)” with j>i, when applied herein to a class ofsubstituents, is meant to refer to embodiments of any of thecompositions, uses, or methods described herein for which each and everyone of the number of carbon members, from i to j including i and j, isindependently realized. By way of example, the term C₁₋₃ refersindependently to embodiments that have one carbon member (C₁),embodiments that have two carbon members (C₂), and embodiments that havethree carbon members (C₃).

The term C_(n-m)alkyl refers to an aliphatic chain, whether straight orbranched, with a total number N of carbon members in the chain thatsatisfies n≤N≤m, with m>n.

Chemical names listed herein were generated using AutoNOM™ software. Ifthere is a discrepancy between a chemical structure and the name listedfor that structure, the structure prevails.

According to the foregoing interpretive considerations on assignmentsand nomenclature, it is understood that explicit reference herein to aset implies, where chemically meaningful and unless indicated otherwise,independent reference to embodiments of such set, and reference to eachand every one of the possible embodiments of subsets of the set referredto explicitly.

In some embodiments, R¹ and R² are each independently —H or alkyl, forexample C₁₋₆alkyl In some embodiments, R¹ and R² are each independently—H or methyl. In some embodiments, R¹ and R² are each independentlyalkyl. In some embodiments, R¹ and R² are both methyl. In someembodiments, R¹ and R² are both —H.

In some embodiments, X is absent. In other embodiments, X is —O—. Insome embodiments, R¹ and R² are each independently alkyl, and X isabsent. In some embodiments, R¹ and R² are both methyl, and X is absent.In other embodiments, R¹ and R² are both —H, and X is —O—. In someembodiments, X is —NR^(z)—, wherein R^(z) is —H or alkyl. In someembodiments, R is —H. In some embodiments, X is R^(z) is alkyl, forexample C₁₋₆alkyl or methyl.

In certain embodiments, R³ is

wherein R¹⁵ and R¹⁶ are each independently —H, —OH, —NH₂, —SH, —N₃,alkyl, alkenyl, alkynyl, -alkyl-OH, -alkyl-NH₂, -alkyl-SH, or -alkyl-N₃.In still other embodiments, R¹⁵ and R¹⁶ are each independently —H,alkyl, —(CH₂)₀₋₆C≡CH, —(CH₂)₀₋₆CH═CH₂, —(CH₂)₀₋₆OH, —(CH₂)₀₋₆NH₂,—(CH₂)₀₋₆SH, or —(CH₂)₀₋₆N₃. In some embodiments, R¹⁵ and R¹⁶ are eachindependently —H, —OH, or alkyl. In some embodiments, R¹⁵ and R¹⁶ areeach independently —H, —OH, or methyl. In some embodiments, R¹⁵ is —OHand R¹⁶ is hydrogen. In some embodiments, R¹⁵ is —OH and R¹⁶ is methyl.

In certain embodiments, R³ is in the R stereochemical configurationrelative to the remainder of the molecule. In other embodiments, R³ isin the S stereochemical configuration relative to the remainder of themolecule. In certain embodiments, the R³ group itself contains one ormore chiral centers, and those stereocenters are each independently inthe R or S configuration.

In certain embodiments, R⁴ is

wherein R¹⁷ and R¹⁸ are each independently —H, —OH, —NH₂, —SH, —N₃,—CO₂H, alkyl, alkenyl, alkynyl, -alkyl-OH, -alkyl-NH₂, -alkyl-SH,-alkyl-N₃ or -alkyl-CO₂H. In other embodiments, R⁴ is

wherein R¹¹ is —H, —OH, —NH₂, —SH, —N₃, —CO₂H, alkyl, alkenyl, alkynyl,-alkyl-OH, -alkyl-NH₂, -alkyl-SH, -alkyl-N₃ or -alkyl-CO₂H, and R¹⁸ is—H, —OH, —NH₂, —SH, —N₃, —CO₂H, alkenyl, alkynyl, -alkyl-OH, -alkyl-NH₂,-alkyl-SH, -alkyl-N₃ or -alkyl-CO₂H. In still other embodiments, R¹ andR¹⁸ are each independently —H, alkyl, —(CH₂)₀₋₆C≡CH, —(CH₂)₀₋₆CH═CH₂,—(CH₂)₀₋₆OH, —(CH₂)₀₋₆NH₂, —(CH₂)₀₋₆SH, or —(CH₂)₀₋₆N₃. In someembodiments, R¹ and R¹⁸ are each independently —H, —OH, —NH₂, —SH, —N₃,—CO₂H, alkyl, -alkyl-NH₂, or -alkyl-N₃. In some embodiments, R¹ and Rare each independently —H, —OH, —NH₂, —SH, —N₃, —CO₂H, methyl, —CH₂NH₂,or —CH₂N₃.

In certain embodiments, R⁴ is taken together with R⁹ and the atoms towhich they are attached to form a substituted or unsubstitutedheterocycloalkyl ring. In certain embodiments, R⁴ is taken together withR⁹ and the atoms to which they are attached to form a 5- to 7-memberheterocycloalkyl ring, which may be unsubstituted or substituted withone or more groups selected from —OH, —NH₂, —SH, and —N₃. In certainembodiments, the heterocycloalkyl ring is a pyrrolidine ring, which maybe unsubstituted or substituted with one or more groups selected from—OH, —NH₂, —SH, and —N₃.

In certain embodiments, R⁴ is in the R stereochemical configurationrelative to the remainder of the molecule. In other embodiments, R⁴ isin the S stereochemical configuration relative to the remainder of themolecule. In certain embodiments, the R⁴ group itself contains one ormore chiral centers, and those stereocenters are each independently inthe R or S configuration.

In certain embodiments, R⁵ is sec-butyl. In other embodiments, R⁵ isisobutyl. In certain embodiments, R⁵ is in the R stereochemicalconfiguration relative to the remainder of the molecule. In otherembodiments, R⁵ is in the S stereochemical configuration relative to theremainder of the molecule. In some embodiments, the chiral center withinthe R⁵ group is in the R configuration, and in other embodiments, thatcenter is in the S configuration.

In certain embodiments, R⁶ is —H. In other embodiments, R⁶ is alkyl, forexample C₁₋₈alkyl, C₁₋₄alkyl, methyl, or ethyl.

In some embodiments, R⁷ and R⁸ are each independently is —H, alkyl,—CO₂R^(a) or —CONR^(b)R^(c); wherein R^(a) is —H or alkyl, for exampleC₁₋₆alkyl or methyl; and R^(b) and R^(c) are each independently —H oralkyl, for example C₁₋₆alkyl or methyl.

In certain embodiments, R⁷ and R⁸ are each independently is substitutedor unsubstituted phenyl or substituted or unsubstituted heterocyclicring, wherein the phenyl or heterocyclic ring may be substituted withone or more groups selected from halo, oxo, hydroxy, amino, alkyl, andalkoxy. In certain other embodiments, R⁷ is unsubstituted 3- to 8-memberheterocyclic ring. In certain other embodiments, R⁷ is substituted 3- to8-member heterocyclic ring. In certain other embodiments, R⁸ is phenylwhich is optionally substituted with halo.

In certain embodiments, R⁷ is in the R stereochemical configurationrelative to the remainder of the molecule. In other embodiments, R⁷ isin the S stereochemical configuration relative to the remainder of themolecule.

In certain embodiments, R⁸ is in the R stereochemical configurationrelative to the remainder of the molecule. In other embodiments, R⁸ isin the S stereochemical configuration relative to the remainder of themolecule.

In some embodiments, R⁷ is —CO₂R^(a)—CONR^(b)R^(c); tetrazolyl orthiazolyl, wherein R^(a) is —H or alkyl, for example C₁₋₆alkyl ormethyl; and R^(b) and R are each independently —H or alkyl, for exampleC₁₋₆alkyl or methyl; and R⁸ is phenyl which is optionally substitutedwith halo.

In some embodiments, R⁹ is —H. In other embodiments, R⁹ is alkyl, forexample C₁₋₈alkyl, C₁₋₄alkyl, methyl, or ethyl. In some embodiments, R⁹is —H or methyl. In some embodiments, R⁹ is methyl.

In some embodiments, R¹⁰ is —H. In other embodiments, R¹⁰ is alkyl, forexample C₁₋₈alkyl, C₁₋₄alkyl, methyl, or ethyl. In some embodiments, R¹⁰is —H or methyl. In some embodiments, R¹⁰ is methyl.

In some embodiments, R¹¹ is —H. In other embodiments, R¹¹ is alkyl, forexample C₁₋₈alkyl, C₁₋₄alkyl, methyl, or ethyl. In some embodiments, R¹¹is —H or methyl. In some embodiments, R¹¹ is methyl.

In some embodiments, R¹² is —H. In other embodiments, R¹² is alkyl, forexample C₁₋₈alkyl, C₁₋₄alkyl, methyl, or ethyl. In some embodiments, R¹is —H or methyl. In some embodiments, R¹² is methyl.

In some embodiments, R¹ is —H. In other embodiments, R¹ is alkyl, forexample C₁₋₈alkyl, C₁₋₄alkyl, methyl, or ethyl. In some embodiments, R¹is —H or methyl. In some embodiments, R¹ is methyl.

In some embodiments, R¹⁴ is —H. In some embodiments, R¹⁴ is alkyl, forexample C₁₋₆alkyl, methyl, or ethyl. In some embodiments, R¹⁴ is —OH.

In certain embodiments, R¹⁴ is in the R stereochemical configurationrelative to the remainder of the molecule. In other embodiments, R¹⁴ isin the S stereochemical configuration relative to the remainder of themolecule.

In some embodiments, R⁷ is —CO₂R^(a), wherein R^(a) is —H or alkyl, forexample C₁₋₆alkyl or methyl; R⁸ is phenyl; and R¹⁴ is —H. In someembodiments, R⁷ is —CONR^(b)R^(c), wherein R^(b) and R^(c) are eachindependently —H or alkyl, for example C₁₋₆alkyl or methyl; R⁸ isphenyl; and R¹⁴ is —H. In some embodiments, R⁷ is alkyl, for exampleC₁₋₆alkyl or methyl; R⁸ is phenyl; and R¹⁴ is —OH. In some embodiments,R⁷ is methyl, R⁸ is phenyl, and R¹⁴ is —OH. In some embodiments, R⁷ andR¹⁴ are both —H, and R⁸ is pyridinyl, piperidinyl, unsubstituted phenyl,or phenyl substituted with halo, for example fluoro, chloro, or bromo.In some embodiments, R⁷ is —CO₂R^(a), wherein R^(a) is —H or alkyl, forexample C₁₋₆alkyl or methyl; R⁸ is —H or alkyl, for example C₁₋₆alkyl ormethyl; and R¹⁴ is alkyl, for example C₁₋₆alkyl, methyl, or ethyl. Insome embodiments, R⁷ is —CO₂R^(a), wherein R^(a) is —H or alkyl, forexample C₁₋₆alkyl or methyl; R⁸ is —H or alkyl, for example C₁₋₆alkyl ormethyl; and R¹⁴ is —OH.

In certain embodiments,

-   R¹ and R² are each independently —H or C₁₋₆alkyl;-   X is —O— or is absent;-   R³ is

-   wherein R¹⁵ and R¹⁶ are each independently —H, —OH, or C₁₋₆alkyl;-   R⁴ is

-   wherein R¹⁷ is —OH, —NH₂, —SH, —N₃, —CO₂H, —C₁₋₆alkyl-NH₂, alkynyl,    alkenyl, or —C₁₋₆alkyl-N₃; and R¹⁸ is —H or C₁₋₆alkyl;-   R⁵ is sec-butyl;-   R⁶ is —H;-   R⁷ is —H, C₁₋₆alkyl, —CO₂R^(a), —CONR^(b)R^(c), tetrazolyl or    thiazolyl; wherein R^(a) is —H or C₁₋₆alkyl; and R^(b) and R^(c) are    each —H or C₁₋₆alkyl;-   R⁸ is —H, C₁₋₆alkyl, substituted or unsubstituted phenyl or    substituted or unsubstituted heterocyclic ring;-   R⁹ is —H;-   R¹⁰, R¹¹, R¹², and R¹³ are each independently C₁₋₆alkyl; and-   R¹⁴ is —H, C₁₋₆alkyl or —OH.

In certain embodiments,

-   R¹ and R² are each independently —H or methyl;-   X is —O— or is absent;-   R³ is

wherein R¹⁵ and R¹⁶ are each independently —H, —OH, or methyl;

-   R⁴ is

wherein R¹⁷ is —OH, —NH₂, —SH, —N₃, —CO₂H, aminomethyl, alkynyl,alkenyl, or azidomethyl; and R¹⁸ is —H or methyl;

-   R⁵ is sec-butyl;-   R⁶ is —H;-   R⁷ is —H, methyl, —CO₂R^(a), or —CONR^(b)R^(c); wherein R^(a) is —H    or methyl; and R^(b) and R^(c) are each —H or methyl;-   R⁸ is —H, methyl, ethyl, pyridinyl, piperidinyl, unsubstituted    phenyl, phenyl substituted with halo;-   R⁹ is —H;-   R¹⁰, R¹¹, R¹², and R¹³ are each methyl; and-   R¹⁴ is —H, methyl or —OH.

In certain embodiments,

-   R¹ and R² are each independently —H or C₁₋₆alkyl;-   X is absent;-   R³ is

wherein R¹⁵ and R¹⁶ are each independently —H, —OH, or C₁₋₆alkyl;

-   R⁴ is

wherein R¹⁷ is —N₃, and R¹⁸ is —H or methyl;

-   R⁵ is sec-butyl;-   R⁶ is —H;-   R⁷ is —H, C₁₋₆alkyl, —CO₂R^(a), —CONR^(b)R^(c), tetrazolyl or    thiazolyl; wherein R^(a) is —H or C₁₋₆alkyl; and R^(b) and R^(c) are    each —H or C₁₋₆alkyl;-   R⁸ is —H, C₁₋₆alkyl, substituted or unsubstituted phenyl or    substituted or unsubstituted heterocyclic ring;-   R⁹ is —H;-   R¹⁰, R¹¹, R¹², and R¹³ are each independently C₁₋₆alkyl; and-   R¹⁴ is —H, C₁₋₆alkyl or —OH.

In certain embodiments,

-   R¹ and R² are each independently —H or C₁₋₆alkyl;-   X is —O—;-   R³ is

wherein R¹⁵ and R¹⁶ are each independently —H, —OH, or C₁₋₆alkyl;

-   R⁴ is

wherein R¹⁷ is —N₃, and R¹⁸ is —H or methyl;

-   R⁵ is sec-butyl;-   R⁶ is —H;-   R⁷ is —H, C₁₋₆alkyl, —CO₂R^(a), —CONR^(b)R^(c), tetrazolyl or    thiazolyl; wherein R^(a) is —H or C₁₋₆alkyl; and R^(b) and R^(c) are    each —H or C₁₋₆alkyl;-   R⁸ is —H, C₁₋₆alkyl, substituted or unsubstituted phenyl or    substituted or unsubstituted heterocyclic ring;-   R⁹ is —H;-   R¹⁰, R¹¹, R¹², and R¹³ are each independently C₁₋₆alkyl; and-   R¹⁴ is —H, C₁₋₆alkyl or —OH.

In some embodiments of Formula (I), wherein,

-   R¹ and R² are each methyl;-   X is absent;-   R³ is a group of the formula:

-   -   wherein R¹⁵ and R¹⁶ are each methyl;

-   R⁴ is a group of the formula:

-   -   wherein R¹⁷ is —N₃, —NH₂, —OH, —SH, and R¹⁸ is —H or methyl;

-   R⁵ is sec-butyl;

-   R⁶ is —H;

-   R⁷ is —CO₂R^(a) or CONR^(b)R^(c),    -   wherein R^(a) is —H or C₁₋₆alkyl;    -   R^(b) and R^(c) are each independently H or C₁₋₆alkyl;

-   R⁸ is phenyl;

-   R⁹ is —H;

-   R¹⁰, R¹¹, R¹², and R¹³ are each independently methyl; and

-   R¹⁴ is —H.

In some embodiments of Formula (I), wherein,

-   R¹ and R² are each —H;-   X is —O—;-   R³ is a group of the formula:

-   -   wherein R¹⁵ and R¹⁶ are each methyl;

-   R⁴ is a group of the formula:

-   -   wherein R¹⁷ is —N₃, and R¹⁸ is —H or methyl;

-   R⁵ is sec-butyl;

-   R⁶ is —H;

-   R⁷ is —CO₂R^(a) or CONR^(b)R^(c),    -   wherein R^(a) is —H or C₁₋₆alkyl;    -   R^(b) and R^(c) are each independently H or C₁₋₆alkyl;

-   R⁸ is phenyl;

-   R⁹ is —H;

-   R¹⁰, R¹¹, R¹², and R¹³ are each independently methyl; and

-   R¹⁴ is —H.

It is to be understood that any variable group definition providedherein can be used in combination with any other variable groupdefinition provided herein, such that all possible combinations andpermutations of variable groups provided herein, where chemicallyfeasible, are contemplated.

In certain embodiments, compounds of Formula (I) are selected from thegroup consisting of:

-   (S)-methyl    2-((2R,3R)-3-((S)-1-((3R,4S,5S)-4-((S)-2-((S)-2-(dimethylamino)-3-methylbutanamido)-3-hydroxy-N-methylpropanamido)-3-methoxy-5-methylheptanoyl)pyrrolidin-2-yl)-3-methoxy-2-methylpropanamido)-3-phenylpropanoate;-   (S)-methyl    2-((2R,3R)-3-((S)-1-((3R,4S,5S)-4-((2S,3R)-2-((S)-2-(dimethylamino)-3-methylbutanamido)-3-hydroxy-N-methylbutanamido)-3-methoxy-5-methylheptanoyl)pyrrolidin    -2-yl)-3-methoxy-2-methylpropanamido)-3-phenylpropanoate;-   (S)-2-(dimethylamino)-N—((S)-3-hydroxy-1-(((3R,4S,5S)-3-methoxy-1-((S)-2-((1R,2R)-1-methoxy-2-methyl-3-oxo-3-((2-(pyridin-2-yl)ethyl)amino)propyl)pyrrolidin-1-yl)-5-methyl-1-oxoheptan-4-yl)(methyl)amino)-1-oxopropan-2-yl)-3-methylbutanamide;-   (2S,3R)-2-((S)-2-(dimethylamino)-3-methylbutanamido)-3-hydroxy-N-((3R,4S,5S)-3-methoxy-1-((S)-2-((1R,2R)-1-methoxy-2-methyl-3-oxo-3-((2-(pyridin-2-yl)ethyl)amino)propyl)pyrrolidin-1-yl)-5-methyl-1-oxoheptan-4-yl)-N-methylbutanamide;-   (2S)-2-(dimethylamino)-N-((2S)-3-hydroxy-1-(((3R,4S,5S)-3-methoxy-1-((2S)-2-((1R,2R)-1-methoxy-2-methyl-3-oxo-3-((2-(piperidin-2-yl)ethyl)amino)propyl)pyrrolidin-1-yl)-5-methyl-1-oxoheptan-4-yl)(methyl)amino)-1-oxopropan-2-yl)-3-methylbutanamide;-   (2S,3R)-2-((S)-2-(dimethylamino)-3-methylbutanamido)-3-hydroxy-N-((3R,4S,5S)-3-methoxy-1-((2S)-2-((1R,2R)-1-methoxy-2-methyl-3-oxo-3-((2-(piperidin-2-yl)ethyl)amino)propyl)pyrrolidin-1-yl)-5-methyl-1-oxoheptan-4-yl)-N-methylbutanamide;-   (S)-methyl    2-((2R,3R)-3-((S)-1-((3R,4S,5S)-4-((2S,3S)-3-azido-2-((S)-2-(dimethylamino)-3-methylbutanamido)-N-methylbutanamido)-3-methoxy-5-methylheptanoyl)pyrrolidin-2-yl)-3-methoxy-2-methylpropanamido)-3-phenylpropanoate;-   (S)-methyl    2-((2R,3R)-3-((S)-1-((3R,4S,5S)-4-((S)-3-amino-2-((S)-2-(dimethylamino)-3-methylbutanamido)-N-methylpropanamido)-3-methoxy-5-methylheptanoyl)pyrrolidin-2-yl)-3-methoxy-2-methylpropanamido)-3-phenylpropanoate;-   (S)—N—((S)-3-amino-1-(((3R,4S,5S)-3-methoxy-1-((S)-2-((1R,2R)-1-methoxy-2-methyl-3-oxo-3-((2-(pyridin-2-yl)ethyl)amino)propyl)pyrrolidin-1-yl)-5-methyl-1-oxoheptan-4-yl)(methyl)amino)-1-oxopropan-2-yl)-2-(dimethylamino)-3-methylbutanamide;-   (S)-methyl    2-((2R,3R)-3-((S)-1-((3R,4S,5S)-4-((S)-3-azido-2-((S)-2-(dimethylamino)-3-methylbutanamido)-N-methylpropanamido)-3-methoxy-5-methylheptanoyl)pyrrolidin-2-yl)-3-methoxy-2-methylpropanamido)-3-phenylpropanoate;-   (S)-methyl    2-((2R,3R)-3-((S)-1-((3R,4S,5S)-4-((S)-4-azido-2-((S)-2-(dimethylamino)-3-methylbutanamido)-N-methylbutanamido)-3-methoxy-5-methylheptanoyl)pyrrolidin-2-yl)-3-methoxy-2-methylpropanamido)-3-phenylpropanoate;-   (S)-methyl    2-((2R,3R)-3-((S)-1-((3R,4S,5S)-4-((S)-4-amino-2-((S)-2-(dimethylamino)-3-methylbutanamido)-N-methylbutanamido)-3-methoxy-5-methylheptanoyl)pyrrolidin-2-yl)-3-methoxy-2-methylpropanamido)-3-phenylpropanoate;-   (S)-2-((S)-2-(aminooxy)-3-methylbutanamido)-N-((3R,4S,5S)-3-methoxy-1-((S)-2-((1R,2R)-1-methoxy-2-methyl-3-oxo-3-((2-(pyridin-2-yl)ethyl)amino)propyl)pyrrolidin-1-yl)-5-methyl-1-oxoheptan-4-yl)-N,3-dimethylbutanamide;-   ((2R,3R)-3-((S)-1-((3R,4S,5S)-4-((S)-2-((S)-2-(dimethylamino)-3-hydroxypropanamido)-N,3-dimethylbutanamido)-3-methoxy-5-methylheptanoyl)pyrrolidin-2-yl)-3-methoxy-2-methylpropanoyl)-L-phenylalanine;-   ((2R,3R)-3-((S)-1-((3R,4S,5S)-4-((S)-2-((2S,3R)-2-(dimethylamino)-3-hydroxybutanamido)-N,3-dimethylbutanamido)-3-methoxy-5-methylheptanoyl)pyrrolidin-2-yl)-3-methoxy-2-methylpropanoyl)-L-phenylalanine;-   (S)-2-((2R,3R)-3-((S)-1-((3R,4S,5S)-4-((2S,3S)-3-azido-2-((S)-2-(dimethylamino)-3-methylbutanamido)-N-methylbutanamido)-3-methoxy-5-methylheptanoyl)pyrrolidin-2-yl)-3-methoxy-2-methylpropanamido)-3-phenylpropanoic    acid;-   (S)—N-((3R,4S,5S)-1-((S)-2-((1R,2R)-3-(((S)-1-amino-1-oxo-3-phenylpropan-2-yl)amino)-1-methoxy-2-methyl-3-oxopropyl)pyrrolidin-1-yl)-3-methoxy-5-methyl-1-oxoheptan-4-yl)-2-((2S,3R)-2-(dimethylamino)-3-hydroxybutanamido)-N,3-dimethylbutanamide;-   (S)—N-((3R,4S,5S)-1-((S)-2-((1R,2R)-3-(((S)-1-amino-1-oxo-3-phenylpropan-2-yl)amino)-1-methoxy-2-methyl-3-oxopropyl)pyrrolidin-1-yl)-3-methoxy-5-methyl-1-oxoheptan-4-yl)-2-((S)-2-(dimethylamino)-3-hydroxypropanamido)-N,3-dimethylbutanamide;-   (S)-methyl    2-((2R,3R)-3-((S)-1-((3R,4S,5S)-4-((R)-2-((S)-2-(dimethylamino)-3-methylbutanamido)-3-mercapto-N-methylpropanamido)-3-methoxy-5-methylheptanoyl)pyrrolidin-2-yl)-3-methoxy-2-methylpropanamido)-3-phenylpropanoate;-   (S)-2-((2R,3R)-3-((S)-1-((3R,4S,5S)-4-((R)-2-((S)-2-(dimethylamino)-3-methylbutanamido)-3-mercapto-N-methylpropanamido)-3-methoxy-5-methylheptanoyl)pyrrolidin-2-yl)-3-methoxy-2-methylpropanamido)-3-phenylpropanoic    acid;-   (S)-2-((2R,3R)-3-((S)-1-((3R,4S,5S)-4-((S)-2-((S)-2-(dimethylamino)-3-methylbutanamido)-3-hydroxy-N-methylpropanamido)-3-methoxy-5-methylheptanoyl)pyrrolidin-2-yl)-3-methoxy-2-methylpropanamido)-3-phenylpropanoic    acid;-   (S)-2-((2R,3R)-3-((S)-1-((3R,4S,5S)-4-((2S,3R)-2-((S)-2-(dimethylamino)-3-methylbutanamido)    -3-hydroxy-N-methylbutanamido)-3-methoxy-5-methylheptanoyl)pyrrolidin-2-yl)-3-methoxy-2-methylpropanamido)-3-phenylpropanoic    acid;-   (S)-methyl    2-((2R,3R)-3-((S)-1-((3R,4S,5S)-4-((S)-2-((S)-2-(dimethylamino)-3-hydroxypropanamido)-N,3-dimethylbutanamido)-3-methoxy-5-methylheptanoyl)pyrrolidin-2-yl)-3-methoxy-2-methylpropanamido)-3-phenylpropanoate;-   (S)-methyl    2-((2R,3R)-3-((S)-1-((3R,4S,5S)-4-((S)-2-((2S,3R)-2-(dimethylamino)-3-hydroxybutanamido)-N,3-dimethylbutanamido)-3-methoxy-5-methylheptanoyl)pyrrolidin-2-yl)-3-methoxy-2-methylpropanamido)-3-phenylpropanoate;-   (S)-methyl    2-((2R,3R)-3-((S)-1-((3R,4S,5S)-4-((2S,3S)-3-amino-2-((S)-2-(dimethylamino)-3-methylbutanamido)-N-methylbutanamido)-3-methoxy-5-methylheptanoyl)pyrrolidin-2-yl)-3-methoxy-2-methylpropanamido)-3-phenylpropanoate;-   (S)-2-((2R,3R)-3-((S)-1-((3R,4S,5S)-4-((2S,3S)-3-amino-2-((S)-2-(dimethylamino)-3-methylbutanamido)-N-methylbutanamido)-3-methoxy-5-methylheptanoyl)pyrrolidin-2-yl)-3-methoxy-2-methylpropanamido)-3-phenylpropanoic    acid;-   (2S,3S)-3-azido-2-((S)-2-(dimethylamino)-3-methylbutanamido)-N-((3R,4S,5S)-3-methoxy-1-((S)-2-((1R,2R)-1-methoxy-2-methyl-3-oxo-3-(phenethylamino)propyl)pyrrolidin-1-yl)-5-methyl-1-oxoheptan-4-yl)-N-methylbutanamide;-   (2S,3S)-3-azido-2-((S)-2-(dimethylamino)-3-methylbutanamido)-N-((3R,4S,5S)-1-((S)-2-((1R,2R)-3-(((1S,2R)-1-hydroxy-1-phenylpropan-2-yl)amino)-1-methoxy-2-methyl-3-oxopropyl)pyrrolidin-1-yl)-3-methoxy-5-methyl-1-oxoheptan-4-yl)-N-methylbutanamide;-   (2S,3S)-3-azido-N-((3R,4S,5S)-1-((S)-2-((1R,2R)-3-((4-chlorophenethyl)amino)-1-methoxy-2-methyl-3-oxopropyl)pyrrolidin-1-yl)-3-methoxy-5-methyl-1-oxoheptan-4-yl)-2-((S)-2-(dimethylamino)-3-methylbutanamido)-N-methylbutanamide;-   (2S,3S)-3-azido-N-((3R,4S,5S)-1-((S)-2-((1R,2R)-3-((2-chlorophenethyl)amino)-1-methoxy-2-methyl-3-oxopropyl)pyrrolidin-1-yl)-3-methoxy-5-methyl-1-oxoheptan-4-yl)-2-((S)-2-(dimethylamino)-3-methylbutanamido)-N-methylbutanamide;-   (2S,3S)-3-amino-2-((S)-2-(dimethylamino)-3-methylbutanamido)-N-((3R,4S,5S)-3-methoxy-1-((S)-2-((1R,2R)-1-methoxy-2-methyl-3-oxo-3-(phenethylamino)propyl)pyrrolidin-1-yl)-5-methyl-1-oxoheptan-4-yl)-N-methylbutanamide;-   (2S,3S)-3-amino-2-((S)-2-(dimethylamino)-3-methylbutanamido)-N-((3R,4S,5S)-1-((S)-2-((1R,2R)-3-(((1S,2R)-1-hydroxy-1-phenylpropan-2-yl)amino)-1-methoxy-2-methyl-3-oxopropyl)pyrrolidin-1-yl)-3-methoxy-5-methyl-1-oxoheptan-4-yl)-N-methylbutanamide;-   (2S,3S)-3-amino-N-((3R,4S,5S)-1-((S)-2-((1R,2R)-3-((4-chlorophenethyl)amino)-1-methoxy-2-methyl-3-oxopropyl)pyrrolidin-1-yl)-3-methoxy-5-methyl-1-oxoheptan-4-yl)-2-((S)-2-(dimethylamino)-3-methylbutanamido)-N-methylbutanamide;-   (2S,3S)-3-amino-N-((3R,4S,5S)-1-((S)-2-((1R,2R)-3-((2-chlorophenethyl)amino)-1-methoxy-2-methyl-3-oxopropyl)pyrrolidin-1-yl)-3-methoxy-5-methyl-1-oxoheptan-4-yl)-2-((S)-2-(dimethylamino)-3-methylbutanamido)-N-methylbutanamide;-   (S)-4-amino-2-((S)-2-(dimethylamino)-3-methylbutanamido)-N-((3R,4S,5S)-3-methoxy-1-((S)-2-((1R,2R)-1-methoxy-2-methyl-3-oxo-3-(phenethylamino)propyl)pyrrolidin-1-yl)-5-methyl-1-oxoheptan-4-yl)-N-methylbutanamide;-   (S)-4-amino-2-((S)-2-(dimethylamino)-3-methylbutanamido)-N-((3R,4S,5S)-1-((S)-2-((1R,2R)-3-(((1S,2R)-1-hydroxy-1-phenylpropan-2-yl)amino)-1-methoxy-2-methyl-3-oxopropyl)pyrrolidin    -1-yl)-3-methoxy-5-methyl-1-oxoheptan-4-yl)-N-methylbutanamide;-   (S)-4-amino-N-((3R,4S,5S)-1-((S)-2-((1R,2R)-3-((4-chlorophenethyl)amino)-1-methoxy-2-methyl-3-oxopropyl)pyrrolidin-1-yl)-3-methoxy-5-methyl-1-oxoheptan-4-yl)-2-((S)-2-(dimethylamino)-3-methylbutanamido)-N-methylbutanamide;-   (S)-4-amino-N-((3R,4S,5S)-1-((S)-2-((1R,2R)-3-((2-chlorophenethyl)amino)-1-methoxy-2-methyl-3-oxopropyl)pyrrolidin-1-yl)-3-methoxy-5-methyl-1-oxoheptan-4-yl)-2-((S)-2-(dimethylamino)-3-methylbutanamido)-N-methylbutanamide;-   methyl    ((2R,3R)-3-((S)-1-((3R,4S,5S)-4-((S)-2-((S)-2-(dimethylamino)-3-methylbutanamido)-N-methylpent    -4-ynamido)-3-methoxy-5-methylheptanoyl)pyrrolidin-2-yl)-3-methoxy-2-methylpropanoyl)-L-phenylalaninate;-   (2S,3S)—N-((3R,4S,5S)-1-((S)-2-((1R,2R)-3-(((S)-1-amino-1-oxo-3-phenylpropan-2-yl)amino)-1-methoxy-2-methyl-3-oxopropyl)pyrrolidin-1-yl)-3-methoxy-5-methyl-1-oxoheptan-4-yl)-3-azido-2-((S)-2-(dimethylamino)-3-methylbutanamido)-N-methylbutanamide;-   (2S,3S)-3-azido-2-((S)-2-(dimethylamino)-3-methylbutanamido)-N-((3R,4S,5S)-3-methoxy-1-((S)-2-((1R,2R)-1-methoxy-2-methyl-3-oxo-3-((2-(pyridin-2-yl)ethyl)amino)propyl)pyrrolidin-1-yl)-5-methyl-1-oxoheptan-4-yl)-N-methylbutanamide;-   (2S,3S)-3-azido-N-((3R,4S,5S)-1-((S)-2-((1R,2R)-3-(((S)-1-(tert-butylamino)-1-oxo-3-phenylpropan-2-yl)amino)-1-methoxy-2-methyl-3-oxopropyl)pyrrolidin-1-yl)-3-methoxy-5-methyl-1-oxoheptan-4-yl)-2-((S)-2-(dimethylamino)-3-methylbutanamido)-N-methylbutanamide;-   methyl    ((2R,3R)-3-((S)-1-((3R,4S,5S)-4-((2S,3S)-3-azido-2-((S)-2-(dimethylamino)-3-methylbutanamido)-N-methylbutanamido)-3-methoxy-5-methylheptanoyl)pyrrolidin-2-yl)-3-methoxy-2-methylpropanoyl)-L-valinate;-   methyl    ((2R,3R)-3-((S)-1-((3R,4S,5S)-4-((S)-6-amino-2-((S)-2-(dimethylamino)-3-methylbutanamido)-N-methylhexanamido)-3-methoxy-5-methylheptanoyl)pyrrolidin-2-yl)-3-methoxy-2-methylpropanoyl)-L-phenylalaninate;-   methyl    ((2R,3R)-3-((S)-1-((3R,4S,5S)-4-((2S,4S)-4-azido-1-(dimethyl-L-valyl)-N-methylpyrrolidine    -2-carboxamido)-3-methoxy-5-methylheptanoyl)pyrrolidin-2-yl)-3-methoxy-2-methylpropanoyl)-L-phenylalaninate;-   (S)-3-((S)-2-(dimethylamino)-3-methylbutanamido)-4-(((3R,4S,5S)-3-methoxy-1-((S)-2-((1R,2R)-1-methoxy-3-(((S)-1-methoxy-1-oxo-3-phenylpropan-2-yl)amino)-2-methyl-3-oxopropyl)pyrrolidin-1-yl)-5-methyl-1-oxoheptan-4-yl)(methyl)amino)-4-oxobutanoic    acid;-   (2S,3R)-2-((S)-2-(dimethylamino)-3-methylbutanamido)-3-hydroxy-N-((3R,4S,5S)-1-((S)-2-((1R,2R)-3-(((1S,2R)-1-hydroxy-1-phenylpropan-2-yl)amino)-1-methoxy-2-methyl-3-oxopropyl)pyrrolidin-1-yl)-3-methoxy-5-methyl-1-oxoheptan-4-yl)-N-methylbutanamide;-   methyl    ((2R,3R)-3-((S)-1-((3R,4S,5S)-4-((S)-2-((S)-2-(dimethylamino)-3-methylbutanamido)-N,3-dimethylbutanamido)-3-methoxy-5-methylheptanoyl)pyrrolidin-2-yl)-3-methoxy-2-methylpropanoyl)-L-serinate;-   methyl    ((2R,3R)-3-((S)-1-((3R,4S,5S)-4-((2S,3S)-3-azido-2-((S)-2-(dimethylamino)-3-methylbutanamido)-N-methylbutanamido)-3-methoxy-5-methylheptanoyl)pyrrolidin-2-yl)-3-methoxy-2-methylpropanoyl)-L-isoleucinate;-   (2S,3S)-3-amino-N-((3R,4S,5S)-1-((S)-2-((1R,2R)-3-(((S)-1-amino-1-oxo-3-phenylpropan-2-yl)amino)-1-methoxy-2-methyl-3-oxopropyl)pyrrolidin-1-yl)-3-methoxy-5-methyl-1-oxoheptan    -4-yl)-2-((S)-2-(dimethylamino)-3-methylbutanamido)-N-methylbutanamide;-   (2S,3S)-3-amino-N-((3R,4S,5S)-1-((S)-2-((1R,2R)-3-(((S)-1-(tert-butylamino)-1-oxo-3-phenylpropan-2-yl)amino)-1-methoxy-2-methyl-3-oxopropyl)pyrrolidin-1-yl)-3-methoxy-5-methyl-1-oxoheptan-4-yl)-2-((S)-2-(dimethylamino)-3-methylbutanamido)-N-methylbutanamide;-   methyl    ((2R,3R)-3-((S)-1-((3R,4S,5S)-4-((S)-3-azido-N-methyl-2-((S)-3-methyl-2-(methylamino)butanamido)propanamido)-3-methoxy-5-methylheptanoyl)pyrrolidin-2-yl)-3-methoxy-2-methylpropanoyl)-L-phenylalaninate;-   methyl    ((2R,3R)-3-((S)-1-((3R,4S,5S)-4-((2S,3S)-3-azido-N-methyl-2-((S)-3-methyl-2-(methylamino)butanamido)butanamido)-3-methoxy-5-methylheptanoyl)pyrrolidin-2-yl)-3-methoxy-2-methylpropanoyl)-L-phenylalaninate;-   (2S,3S)—N-((3R,4S,5S)-1-((S)-2-((1R,2R)-3-(((S)-1-amino-1-oxo-3-phenylpropan-2-yl)amino)-1-methoxy-2-methyl-3-oxopropyl)pyrrolidin-1-yl)-3-methoxy-5-methyl-1-oxoheptan-4-yl)-3-azido-N-methyl-2-((S)-3-methyl-2-(methylamino)butanamido)butanamide;-   ((2S,3S)-3-azido-N-((3R,4S,5S)-1-((S)-2-((1R,2R)-3-(((S)-1-(tert-butylamino)-1-oxo-3-phenylpropan-2-yl)amino)-1-methoxy-2-methyl-3-oxopropyl)pyrrolidin-1-yl)-3-methoxy-5-methyl-1-oxoheptan-4-yl)-N-methyl-2-((S)-3-methyl-2-(methylamino)butanamido)butanamide;-   tert-butyl    ((2R,3R)-3-((S)-1-((3R,4S,5S)-4-((2S,3S)-3-azido-N-methyl-2-((S)-3-methyl-2-(methylamino)butanamido)butanamido)-3-methoxy-5-methylheptanoyl)pyrrolidin-2-yl)-3-methoxy-2-methylpropanoyl)-L-phenylalaninate;-   ((2R,3R)-3-((S)-1-((3R,4S,5S)-4-((2S,3S)-3-azido-N-methyl-2-((S)-3-methyl-2-(methylamino)butanamido)butanamido)-3-methoxy-5-methylheptanoyl)pyrrolidin-2-yl)-3-methoxy-2-methylpropanoyl)-L-phenylalanine;-   tert-butyl    ((2R,3R)-3-((S)-1-((3R,4S,5S)-4-((2S,3S)-3-azido-2-((S)-2-(dimethylamino)-3-methylbutanamido)-N-methylbutanamido)-3-methoxy-5-methylheptanoyl)pyrrolidin-2-yl)-3-methoxy-2-methylpropanoyl)-L-phenylalaninate;-   (2S,3S)-3-azido-2-((S)-2-(dimethylamino)-3-methylbutanamido)-N-((3R,4S,5S)-3-methoxy-1-((S)-2-((1R,2R)-1-methoxy-2-methyl-3-oxo-3-(((S)-2-phenyl-1-(1H-tetrazol-5-yl)ethyl)amino)propyl)pyrrolidin-1-yl)-5-methyl-1-oxoheptan-4-yl)-N-methylbutanamide;-   (2S,3S)-3-azido-N-((3R,4S,5S)-3-methoxy-1-((S)-2-((1R,2R)-1-methoxy-2-methyl-3-oxo-3-(((S)    -2-phenyl-1-(1H-tetrazol-5-yl)ethyl)amino)propyl)pyrrolidin-1-yl)-5-methyl-1-oxoheptan-4-yl)-N-methyl    -2-((S)-3-methyl-2-(methylamino)butanamido)butanamide;-   (2S,3S)-3-azido-2-((S)-2-(dimethylamino)-3-methylbutanamido)-N-((3R,4S,5S)-3-methoxy-1-((S)-2-((1R,2R)-1-methoxy-2-methyl-3-oxo-3-(((S)-2-phenyl-1-(thiazol-2-yl)ethyl)amino)propyl)pyrrolidin-1-yl)-5-methyl-1-oxoheptan-4-yl)-N-methylbutanamide;-   tert-butyl    ((2R,3R)-3-((S)-1-((3R,4S,5S)-4-((2S,3S)-3-amino-2-((S)-2-(dimethylamino)-3-methylbutanamido)-N-methylbutanamido)-3-methoxy-5-methylheptanoyl)pyrrolidin-2-yl)-3-methoxy-2-methylpropanoyl)-L-phenylalaninate;-   (2S,3S)-3-amino-2-((S)-2-(dimethylamino)-3-methylbutanamido)-N-((3R,4S,5S)-3-methoxy-1-((S)-2-((1R,2R)-1-methoxy-2-methyl-3-oxo-3-(((S)-2-phenyl-1-(1H-tetrazol-5-yl)ethyl)amino)propyl)pyrrolidin-1-yl)-5-methyl-1-oxoheptan-4-yl)-N-methylbutanamide;    and-   (2S,3S)-3-amino-2-((S)-2-(dimethylamino)-3-methylbutanamido)-N-((3R,4S,5S)-3-methoxy-1-((S)-2-((1R,2R)-1-methoxy-2-methyl-3-oxo-3-(((S)-2-phenyl-1-(thiazol-2-yl)ethyl)amino)propyl)pyrrolidin-1-yl)-5-methyl-1-oxoheptan-4-yl)-N-methylbutanamide;    and pharmaceutically acceptable salts thereof.

Also provided herein are pharmaceutically acceptable salts of thecompounds of Formula (I), preferably of those described above and thespecific compounds exemplified herein, pharmaceutical compositionscomprising such salts, and methods of using such salts.

A “pharmaceutically acceptable salt” is intended to mean a salt of afree acid or base of a compound represented herein that is non-toxic,biologically tolerable, or otherwise biologically suitable foradministration to the subject. See, generally, S. M. Berge, et al.“Pharmaceutical Salts,” J. Pharm. Sci. 1977, 66, 1-19. Preferredpharmaceutically acceptable salts are those that are pharmacologicallyeffective and suitable for contact with the tissues of subjects withoutundue toxicity, irritation, or allergic response. A compound describedherein may possess a sufficiently acidic group, a sufficiently basicgroup, or both types of functional groups, and accordingly react with anumber of inorganic or organic bases, and inorganic and organic acids,to form a pharmaceutically acceptable salt. Examples of pharmaceuticallyacceptable salts include acid addition salts such as sulfates,pyrosulfates, bisulfates, sulfites, bisulfites, phosphates,monohydrogen-phosphates, dihydrogenphosphates, metaphosphates,pyrophosphates, chlorides, bromides, iodides, acetates, propionates,decanoates, caprylates, acrylates, formates, isobutyrates, caproates,heptanoates, propiolates, oxalates, malonates, succinates, suberates,sebacates, fumarates, maleates, butyne-1,4-dioates, hexyne-1,6-dioates,benzoates, chlorobenzoates, methylbenzoates, dinitrobenzoates,hydroxybenzoates, methoxybenzoates, phthalates, sulfonates,methylsulfonates, propylsulfonates, besylates, xylenesulfonates,naphthalene-1-sulfonates, naphthalene-2-sulfonates, phenylacetates,phenylpropionates, phenylbutyrates, citrates, lactates,γ-hydroxybutyrates, glycolates, tartrates, and mandelates, and saltswith inorganic bases such as sodium, potassium, magnesium, calcium,aluminum, and the like or organic bases such as methylamine, ethylamine,ethanolamine, lysine, ornithine, and the like, salts with various aminoacids or amino acid derivatives such as acetylleucine and the like,ammonium salts, etc.

For treatment purposes, pharmaceutical compositions comprising compoundsdescribed herein may further comprise one or morepharmaceutically-acceptable excipients. A pharmaceutically-acceptableexcipient is a substance that is non-toxic and otherwise biologicallysuitable for administration to a subject. Such excipients facilitateformulation and administration of a compound described herein and arecompatible with the active ingredient. Examples ofpharmaceutically-acceptable excipients include stabilizers, lubricants,surfactants, diluents, anti-oxidants, binders, coloring agents,emulsifiers, or taste-modifying agents. In preferred embodiments,pharmaceutical compositions are sterile compositions.

The pharmaceutical compositions described herein may be formulated assolutions, emulsions, suspensions, or dispersions in suitablepharmaceutical solvents or carriers, or as pills, tablets, lozenges,suppositories, powders for reconstitution, or capsules along with solidcarriers according to conventional methods known in the art forpreparation of various dosage forms. For topical applications, thecompounds described herein are preferably formulated as creams orointments or a similar vehicle suitable for topical administration. Thepharmaceutical compositions and compounds described herein may beadministered in the inventive methods by a suitable route of delivery,e.g., oral, nasal, parenteral, rectal, topical, ocular, or byinhalation.

The term “treat” or “treating” as used herein is intended to refer toadministration of a compound described herein to a subject for thepurpose of creating a therapeutic benefit.

Treating includes reversing, ameliorating, alleviating, inhibiting theprogress of, or lessening the severity of, a disease, disorder, orcondition, or one or more symptoms of cancer. The term “subject” refersto a mammalian patient in need of such treatment, such as a human.

In treatment methods provided herein, “an effective amount” means anamount or dose sufficient to generally bring about the desiredtherapeutic benefit in subjects needing such treatment. Effectiveamounts or doses of the compounds described herein may be ascertained byroutine methods, such as modeling, dose escalation or clinical trials,taking into account routine factors, e.g., the mode or route ofadministration or drug delivery, the pharmacokinetics of the agent, theseverity and course of the infection, the subject's health status,condition, and weight, and the judgment of the treating physician. Anexemplary dose is in the range of about 1 ug to 2 mg of active compoundper kilogram of subject's body weight per day, preferably about 0.05 to100 mg/kg/day, or about 1 to 35 mg/kg/day, or about 0.1 to 10 mg/kg/day.The total dosage may be given in single or divided dosage units (e.g.,BID, TID, QID).

The compounds described herein may be used in pharmaceuticalcompositions or methods in combination with additional activeingredients in the treatment of cancer. The additional activeingredients may be administered separately from a compound describedherein or may be included with a compound described herein in apharmaceutical composition provided herein. For example, additionalactive ingredients are those that are known or discovered to beeffective in treating cancer, including those active against anothertarget associated with cancer, such as but not limited to, Velcade,Rituximab, Methotrexate, Herceptin, Vincristine, Prednisone, Irinotecan,or the like, or a combination thereof. Such a combination may serve toincrease efficacy, decrease one or more side effects, or decrease therequired dose of a disclosed compound.

The compounds described herein may be used in pharmaceuticalcompositions or methods in combination with additional activeingredients in the treatment of cancer. The additional activeingredients may be administered separately from a compound describedherein or may be included with a compound described herein in apharmaceutical composition provided herein. For example, additionalactive ingredients are those that are known or discovered to beeffective in treating cancer, including those active against anothertarget associated with cancer, such as but not limited to, Velcade,Rituximab, Methotrexate, Herceptin, Vincristine, Prednisone, Irinotecan,or the like, or a combination thereof. Such a combination may serve toincrease efficacy, decrease one or more side effects, or decrease therequired dose of a disclosed compound.

Compounds of Formula (I) will now be described by reference toillustrative synthetic schemes for their general preparation below andthe specific examples that follow. Artisans will recognize that, toobtain the various compounds herein, starting materials may be suitablyselected so that the ultimately desired substituents will be carriedthrough the reaction scheme with or without protection as appropriate toyield the desired product. Alternatively, it may be necessary ordesirable to employ, in the place of the ultimately desired substituent,a suitable group that may be carried through the reaction scheme andreplaced as appropriate with the desired substituent. In addition, oneof skill in the art will recognize that protecting groups may be used toprotect certain functional groups (amino, carboxy, or side chain groups)from reaction conditions, and that such groups are removed understandard conditions when appropriate. Each of the reactions depicted inScheme A is preferably run at a temperature from about room temperatureto the reflux temperature of the organic solvent used. Unless otherwisespecified, the variables are as defined above in reference to Formula(I).

Referring to Scheme A, the preparation of compounds of Formula (I)begins with a protected acid form of dolaisoleuine (Dil) labeled (A)(see Pettit et al. (1994) J. Org. Chem. 59:1796-1800). Compound (A) isdepicted with a tert-butyl ester protecting group, but one of skill inthe art may select an appropriate replacement. Coupling with anitrogen-protected valine or isoleucine derivative (B), where PG is asuitable amino protecting group such as a Boc (t-butoxycarbonyl) orfluorenylmethyloxycarbonyl (Fmoc) group, is effected under standardpeptide coupling conditions. For example, reactions are run in thepresence of diethyl cyanophosphonate (DEPC), PyBrOP, PyBOP, BOP,diisopropylcarbodiimide (DIC), dicyclohexylcarbodiimide (DCC),1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDCI),1-hydroxybenzotriazole (HOBt), 1-hydroxy-7-aza-benzotriazole (HOAt),HBTU (O-benzotriazol-1-yl-N,N,N′,N′-tetramethyluroniumhexafluorophosphate), HATU(O-(7-azabenzotriazol-1-yl)-1,1,3,3-tetramethyluroniumhexafluorophosphate), and the like, or a combination thereof. Reactionsare typically run in the presence of a tertiary amine base, such asdiisopropylethylamine. Suitable solvents include dichloromethane,N,N-dimethylformamide (DMF), dimethyl sulfoxide (DMSO), ethyl acetateand the like. The amino protecting group on resultant dipeptide (C) isremoved by deprotection under suitable conditions. For example, where PGis a Boc group, compound (C) is treated with trifluoroacetic acid toform free amine (D). Where PG is an Fmoc group, compound (C) is treatedwith piperidine or diethylamine to yield compound (D). Compound (D) isthen coupled to amino acid derivative (E), in protected form ifnecessary, under peptide coupling conditions as described above, togenerate tripeptide (F). Treatment with acid removes the carboxyprotecting group to provide free acid (G).

Referring to Scheme B, the amino-protected dolaproine (Dap) designatedas (H) (see Pettit et al. (1994) J. Org. Chem. 59:6287-6295) is coupledwith amine (J) (which is prepared using methods known to one in the art)under peptide coupling conditions as described above. Resultingdipeptide (K) is deprotected as discussed for Scheme A to providecompound (L).

Referring to Scheme C, acid (G) and amine (L) are coupled under peptidecoupling conditions as discussed above to provide compounds of Formula(I). Where the result of the reaction is a protected form of Formula(I), suitable deprotection conditions are employed to give the targetcompound.

EXAMPLES

The following examples are offered to illustrate but not to limit thecompositions, uses, and methods provided herein. The compounds areprepared using the general methods described above.

The following chemical abbreviations are used throughout the Examples:Dov (dolavaline); Abu (2-aminobutyric acid); Dil (dolaisoleuine); Dpr(2,3-diaminopropionic acid); Su (succinimidinyl); Dab(2,4-diaminobutyric acid); Dap (dolaproine); Bzl (benzyl); and Tr(trityl).

LCMS retention times were acquired on an Aquity UPLC BeH C8 1.7 μm2.1×50 mm column, 40° C., using one of the following methods (asindicated):

Method A: 0-0.50 min: isocratic 80 water/10 acetonitrile/10 1% formicacid in water; 0.50-3.50 min: linear gradient 80 water/10acetonitrile/10 1% formic acid in water to 0 water/90 acetonitrile/10 1%formic acid in water; 3.50-3.99 min isocratic 0 water/90 acetonitrile/101% formic acid in water; 3.99-4.00 min linear gradient 0 water/90acetonitrile/10 1% formic acid in water to 80 water/10 acetonitrile/101% formic acid in water.

Method B: 0-0.50 min: isocratic 85 water/5 acetonitrile/10 1% formicacid in water; 0.50-1.60 min: linear gradient 85 water/5 acetonitrile/101% formic acid in water to 0 water/98 acetonitrile/2 1% formic acid inwater; 1.60-1.80 min isocratic 0 water/98 acetonitrile/2 1% formic acidin water; 1.80-1.90 min linear gradient 0 water/98 acetonitrile/2 1%formic acid in water to 85 water/5 acetonitrile/10 1% formic acid inwater; 1.90-2.00 min isocratic 85 water/5 acetonitrile/10 1% formic acidin water.

Example 1 (S)-methyl2-((2R,3R)-3-((S)-1-((3R,4S,5S)-4-((S)-2-((S)-2-(dimethylamino)-3-methylbutanamido)-3-hydroxy-N-methylpropanamido)-3-methoxy-5-methylheptanoyl)pyrrolidin-2-yl)-3-methoxy-2-methylpropanamido)-3-phenylpropanoate

To a stirred room temperature suspension of Boc-Dap-OH dicyclohexylaminesalt (8.00 g, 17.1 mmol) and H-Phe-OMe HCl salt (4.42 g, 20.5 mmol) inCH₂Cl₂ (20 mL) was added diisopropylethylamine (DIEA; 9.13 mL, 51.3mmol), followed by diethylpyrocarbonate (DEPC; 5.15 mL, 34.2 mmol).After 10 h, analysis by liquid chromatography/mass spectrometry (LCMS)showed the reaction was complete. Boc-Dap-Phe-OMe was isolated by flashchromatography on silica gel (silica gel 40 μm, 60 Å, 3.0×17.0 cm) using2% to 10% MeOH in CH₂Cl₂ as the eluent. A total of 7.45 g ofBoc-Dap-Phe-OMe (16.61 mmol, 97% yield) was obtained.

To a stirred room temperature solution of Boc-Dap-Phe-OMe (4.67 g, 10.4mmol) in CH₂Cl₂ (10 mL) was added trifluoroacetic acid (TFA; 10 mL).After 10 h, analysis by LCMS showed the reaction was complete. The crudeoil was purified by preparatory RP-HPLC with a Phenomenex Synergi 10μMax-RP 80 Å column (150×30 mm) using 10% to 90% MeCN in 0.05% aqueousTFA as the eluent. A total of 2.52 g H-Dap-Phe-OMe (6.23 mmol, 59%) wasobtained as the TFA salt.

To a stirred room temperature solution of Fmoc-Ser(Bzl)-OH (2.82 g, 6.76mmol) and H-Dil-OtBu hydrochloride (2.00 g, 6.76 mmol) in ethyl acetate(EtOAc; 15 mL) was added DIEA (2.17 mL, 12.2 mmol). The solution wascooled to (0° C.) and stirred for 20 min. DIEA (2.17 mL, 12.2 mmol) wasadded to the reaction mixture. The solution was cooled to (0° C.) andstirred for 20 min. 2-Chloro-1-methylpyridinium iodide (CMPI; 2.76 g,10.8 mmol) was added to the reaction mixture and the reaction mixturewas allowed to reach room temperature. After 12 h, analysis by LCMSshowed the reaction was complete. The crude reaction was washed with 0.1M HCl (150 mL×2). The organic fraction was dried over a pad of magnesiumsulfate, filtered and concentrated in vacuo. Fmoc-Ser(Bzl)-Dil-OtBu wasisolated by flash chromatography on silica gel (silica gel 40 μm, 60 Å,3.0×17.0 cm) using 18% to 90% EtOAc in hexanes as the eluent. A total of3.75 g of Fmoc-Ser(Bzl)-Dil-OtBu (5.69 mmol, 84% yield) was obtained.

To a stirred room temperature solution of Fmoc-Ser(Bzl)-Dil-OtBu (1.79g, 2.72 mmol) in MeCN (5 mL) was added piperidine (4 mL). After 5 h,analysis by LCMS showed the reaction was complete. The crude reactionmixture was extracted with Hexanes and the MeCN layer was concentratedin vacuo to yield crude H-Ser(Bzl)-Dil-OtBu that was used withoutfurther purification.

To a stirred room temperature suspension of crude H-Ser(Bzl)-Dil-OtBuand Dov (0.790 g, 5.44 mmol) in DMF (10 mL) was added DIEA (1.45 mL,8.16 mmol), followed by HATU (2.07 g, 5.44 mmol). After 5 h, analysis byLCMS showed that the reaction was complete. The crude reaction mixturewas diluted with saturated sodium bicarbonate (10 mL) and extracted withEtOAc (40 mL×2). The combined organic fractions were washed with brine,dried over a pad of magnesium sulfate, filtered, and concentrated invacuo. The crude oil was purified by preparatory RP-HPLC with aPhenomenex Synergi 10μ Max-RP 80 Å column (150×30 mm) using 10% to 90%MeCN in 0.05% aqueous TFA as the eluent. A total of 0.882 gDov-Ser(Bzl)-Dil-OtBu (1.30 mmol, 48%) was obtained as the TFA salt.

To a room temperature solution of Dov-Ser(Bzl)-Dil-OtBu (0.288 g, 0.425mmol) in CH₂Cl₂ (5 mL) was added TFA (4 mL). After 10 h, analysis byLCMS showed the reaction was complete. Volatile organics were evaporatedin vacuo to yield crude Dov-Ser(Bzl)-Dil-OH TFA salt that was usedwithout further purification.

To a stirred room temperature suspension of crude Dov-Ser(Bzl)-Dil-OHTFA salt and H-Dap-Phe-OMe TFA salt (0.163 g, 0.467 mmol) in DMF (10 mL)was added DIEA (0.303 mL, 1.70 mmol), followed by HATU (0.323 g, 0.850mmol). After 6 h, analysis by LCMS showed the reaction was complete. Thecrude reaction mixture was diluted with saturated sodium bicarbonate (10mL) and extracted with EtOAc (40 mL×2). The combined organic fractionswere washed with brine, dried over a pad of magnesium sulfate, filtered,and concentrated in vacuo. The crude oil was purified by preparatoryRP-HPLC with a Phenomenex Synergi 10μ Max-RP 80 Å column (150×30 mm)using 10% to 90% MeCN in 0.05% aqueous TFA as the eluent. A total of0.217 g Dov-Ser(Bzl)-Dil-Dap-Phe-OMe (0.228 mmol, 54%) was obtained asthe TFA salt.

A stirred room temperature suspension of Dov-Ser(Bzl)-Dil-Dap-Phe-OMeTFA salt (0.217 g, 0.228 mmol) and palladium on activated charcoal (10%Pd basis, 0.174 g) in MeOH (5 mL) was hydrogenated under refluxingconditions. After 48 h, analysis by LCMS showed the reaction wascomplete. The crude reaction mixture was filtered over a pad ofdiatomaceous earth and the filtrate was concentrated. The crude oil waspurified by preparatory RP-HPLC with a Phenomenex Synergi 10μ Max-RP 80Å column (150×30 mm) using 10% to 90% MeCN in 0.05% aqueous TFA as theeluent. A total of 0.104 g of the title compound was obtained as the TFAsalt (0.121 mmol, 47%). LCMS RT=2.32 min (Method A); ESI-MS m/z 748.72[M+H]+; HRMS m/z 748.4846 [C₃₉H₆₅N₅O₉+H]⁺.

Example 2 (S)-methyl2-((2R,3R)-3-((S)-1-((3R,4S,5S)-4-((2S,3R)-2-((S)-2-(dimethylamino)-3-methylbutanamido)-3-hydroxy-N-methylbutanamido)-3-methoxy-5-methylheptanoyl)pyrrolidin-2-yl)-3-methoxy-2-methylpropanamido)-3-phenylpropanoate

To a stirred room temperature solution of Fmoc-Thr(Bzl)-OH (2.92 g, 6.76mmol) and H-Dil-OtBu hydrochloride (2.00 g, 6.76 mmol) in EtOAc (15 mL)was added DIEA (2.17 mL, 12.2 mmol). The solution was cooled (0° C.) andstirred for 20 min. DIEA (2.17 mL, 12.2 mmol) was added to the reactionmixture. The solution was cooled (0° C.) and stirred for 20 min. CMPI(2.76 g, 10.8 mmol) was added to the reaction mixture and the reactionmixture was allowed to reach room temperature. After 12 h, analysis byLCMS showed the reaction was complete. The crude reaction was washedwith 0.1 M HCl (100 mL×2). The organic fraction was dried over a pad ofmagnesium sulfate, filtered and concentrated in vacuo.Fmoc-Thr(Bzl)-Dil-OtBu was isolated by flash chromatography on silicagel (silica gel 40 μm, 60 Å, 3.0×17.0 cm) using 18% to 90% EtOAc inhexanes as the eluent. A total of 3.71 g of Fmoc-Thr(Bzl)-Dil-OtBu (5.51mmol, 82% yield) was obtained.

To a stirred room temperature solution of Fmoc-Thr(Bzl)-Dil-OtBu (3.71g, 5.51 mmol) in MeCN (5 mL) was added piperidine (4 mL). After 5 h,analysis by LCMS showed the reaction was complete. The crude reactionmixture was extracted with Hexanes and the MeCN layer was concentratedin vacuo to yield crude H-Thr(Bzl)-Dil-OtBu that was used withoutfurther purification.

To a stirred room temperature suspension of crude H-Thr(Bzl)-Dil-OtBuand Dov (1.60 g, 11.0 mmol) in DMF (20 mL) was added DIEA (2.95 mL, 16.5mmol), followed by HATU (4.19 g, 11.0 mmol). After 6 h, analysis by LCMSshowed that the reaction was complete. The crude reaction mixture wasdiluted with saturated sodium bicarbonate (10 mL) and extracted withEtOAc (40 mL×2). The combined organic fractions were washed with brine,dried over a pad of magnesium sulfate, filtered, and concentrated invacuo. The crude oil was purified by preparatory RP-HPLC with aPhenomenex Synergi 10μ Max-RP 80 Å column (150×30 mm) using 10% to 90%MeCN in 0.05% aqueous TFA as the eluent. A total of 1.09 gDov-Ser(Bzl)-Dil-OtBu (1.58 mmol, 29%) was obtained as the TFA salt.

To a room temperature solution of Dov-Thr(Bzl)-Dil-OtBu TFA salt (0.331g, 0.478 mmol) in CH₂Cl₂ (5 mL) was added TFA (4 mL). After 10 h,analysis by LCMS showed the reaction was complete. Volatile organicswere evaporated in vacuo to yield crude Dov-Thr(Bzl)-Dil-OH TFA saltthat was used without further purification.

To a stirred room temperature suspension of crude Dov-Thr(Bzl)-Dil-OHTFA salt and H-Dap-Phe-OMe TFA salt (0.183 g, 0.526 mmol) in DMF (10 mL)was added DIEA (0.341 mL, 1.92 mmol), followed by HATU (0.364 g, 0.957mmol). After 24 h, analysis by LCMS showed the reaction was complete.The crude reaction mixture was diluted with saturated sodium bicarbonate(10 mL) and extracted with EtOAc (40 mL×2). The combined organicfractions were washed with brine, dried over a pad of magnesium sulfate,filtered, and concentrated in vacuo. The crude oil was purified bypreparatory RP-HPLC with a Phenomenex Synergi 10μ Max-RP 80 Å column(150×30 mm) using 10% to 90% MeCN in 0.05% aqueous TFA as the eluent. Atotal of 0.290 g Dov-Thr(Bzl)-Dil-Dap-Phe-OMe (0.300 mmol, 63%) wasobtained as the TFA salt.

A stirred room temperature suspension of Dov-Thr(Bzl)-Dil-Dap-Phe-OMeTFA salt (0.290 g, 0.300 mmol) and palladium on activated charcoal (10%Pd basis, 0.232 g) in MeOH (5 mL) was hydrogenated. After 24 h, analysisby LCMS showed the reaction was complete. The crude reaction mixture wasfiltered over a pad of diatomaceous earth and the filtrate wasconcentrated. The crude oil was purified by preparatory RP-HPLC with aPhenomenex Synergi 10μ Max-RP 80 Å column (150×30 mm) using 10% to 90%MeCN in 0.05% aqueous TFA as the eluent. A total of 0.129 g of the titlecompound was obtained as the TFA salt (0.147 mmol, 43%). LCMS RT=2.40min (Method A); ESI-MS m/z 762.75 [M+H]⁺; HRMS m/z 762.5009[C₄₀H₆₇N₅O₉+H]⁺.

Example 3(S)-2-(dimethylamino)-N—((S)-3-hydroxy-1-(((3R,4S,5S)-3-methoxy-1-((S)-2-((1R,2R)-1-methoxy-2-methyl-3-oxo-3-((2-(pyridin-2-yl)ethyl)amino)propyl)pyrrolidin-1-yl)-5-methyl-1-oxoheptan-4-yl)(methyl)amino)-1-oxopropan-2-yl)-3-methylbutanamide

To a stirred room temperature suspension of Boc-Dap-OH dicyclohexylaminesalt (10.0 g, 21.4 mmol) and 2-(2-pyridyl)ethylamine (3.83 mL, 32.0mmol) in CH₂Cl₂ (20 mL) was added DIEA (11.4 mL, 64.1 mmol), followed byDEPC (4.83 mL, 32.0 mmol). After overnight stirring, analysis by LCMSshowed the reaction was complete. Boc-Dap-2-(2-pyridyl)ethylamine wasisolated by flash chromatography on silica gel (silica gel 40 μm, 60 Å,3.0×17.0 cm) using 2% to 10% MeOH/1% NEt₃ in CH₂Cl₂ as the eluent. Atotal of 7.42 g of Boc-Dap-2-(2-pyridyl)ethylamine (19.0 mmol, 89%yield) was obtained.

To a stirred room temperature solution ofBoc-Dap-2-(2-pyridyl)ethylamine (7.42 g, 19.0 mmol) in CH₂Cl₂ (10 mL)was added TFA (10 mL). After overnight stirring, analysis by LCMS showedthe reaction was complete. The crude oil was purified by preparatoryRP-HPLC with a Phenomenex Synergi 10μ Max-RP 80 Å column (150×30 mm)using 10% to 90% MeCN in 0.05% aqueous TFA as the eluent. A total of4.50 g H-Dap-(2-pyridyl)ethylamine (11.1 mmol, 59%) was obtained as theTFA salt.

To a room temperature solution of Dov-Ser(Bzl)-Dil-OtBu (0.287 g, 0.423mmol) in CH₂Cl₂ (5 mL) was added TFA (4 mL). After 10 h, analysis byLCMS showed the reaction was complete. Volatile organics were evaporatedin vacuo to yield crude Dov-Ser(Bzl)-Dil-OH TFA salt that was usedwithout further purification.

To a stirred room temperature suspension of crude Dov-Ser(Bzl)-Dil-OHTFA salt and H-Dap-2-(2-pyridyl)ethylamine TFA salt (0.136 g, 0.466mmol) in DMF (10 mL) was added DIEA (0.302 mL, 1.70 mmol) followed byHATU (0.322 g, 0.847 mmol). After 6 h, analysis by LCMS showed thereaction was complete. The crude reaction mixture was diluted withsaturated sodium bicarbonate (10 mL) and extracted with EtOAc (40 mL×2).The combined organic fractions were washed with brine, dried over a padof magnesium sulfate, filtered, and concentrated in vacuo. The crude oilwas purified by preparatory RP-HPLC with a Phenomenex Synergi 10μ Max-RP80 Å column (150×30 mm) using 10% to 90% MeCN in 0.05% aqueous TFA asthe eluent. A total of 0.258 gDov-Ser(Bzl)-Dil-Dap-2-(2-pyridyl)ethylamine (0.288 mmol, 68%) wasobtained as the TFA salt.

A stirred room temperature suspension ofDov-Ser(Bzl)-Dil-Dap-2-(2-pyridyl)ethylamine TFA salt (0.258 g, 0.330mmol), ammonium formate (0.062 g, 0.991 mmol), and palladium onactivated charcoal (10% Pd basis, 0.100 g) in MeOH (5 mL) washydrogenated. After 12 h, analysis by LCMS showed the reaction wascomplete. The crude reaction mixture was filtered over a pad ofdiatomaceous earth and the filtrate was concentrated. The crude oil waspurified by preparatory RP-HPLC with a Phenomenex Synergi 10μ Max-RP 80Å column (150×30 mm) using 10% to 90% MeCN in 0.05% aqueous TFA as theeluent. A total of 0.014 g of the title compound was obtained as the TFAsalt (0.017 mmol, 5%). LCMS RT=1.76 min (Method A); ESI-MS m/z 691.56[M+H]⁺; HRMS m/z 691.4755 [C₃₆H₆₂N₆O₇+H]⁺.

Example 4(2S,3R)-2-((S)-2-(dimethylamino)-3-methylbutanamido)-3-hydroxy-N-((3R,4S,5S)-3-methoxy-1-((S)-2-((1R,2R)-1-methoxy-2-methyl-3-oxo-3-((2-(pyridin-2-yl)ethyl)amino)propyl)pyrrolidin-1-yl)-5-methyl-1-oxoheptan-4-yl)-N-methylbutanamide

To a room temperature solution of Dov-Thr(Bzl)-Dil-OtBu (0.357 g, 0.618mmol) in CH₂Cl₂ (5 mL) was added TFA (4 mL). After 10 h, analysis byLCMS showed the reaction was complete. Volatile organics were evaporatedin vacuo to yield crude Dov-Thr(Bzl)-Dil-OH TFA salt that was usedwithout further purification.

To a stirred room temperature suspension of crude Dov-Thr(Bzl)-Dil-OHTFA salt and H-Dap-2-(2-pyridyl)ethylamine TFA salt (0.198 g, 0.680mmol) in DMF (10 mL) was added DIEA (0.441 mL, 2.47 mmol), followed byHATU (0.470 g, 1.24 mmol) and HOBt (0.189 g, 1.24 mmol). After 6 h,analysis by LCMS showed the reaction was complete. The crude reactionmixture was diluted with saturated sodium bicarbonate (10 mL) andextracted with EtOAc (40 mL×2). The combined organic fractions werewashed with brine, dried over a pad of magnesium sulfate, filtered, andconcentrated in vacuo. The crude oil was purified by preparatory RP-HPLCwith a Phenomenex Synergi 10μ Max-RP 80 Å column (150×30 mm) using 10%to 90% MeCN in 0.05% aqueous TFA as the eluent. A total of 0.356 gDov-Thr(Bzl)-Dil-Dap -2-(2-pyridyl)ethylamine (0.392 mmol, 63%) wasobtained as the TFA salt.

A stirred room temperature suspension ofDov-Thr(Bzl)-Dil-Dap-2-(2-pyridyl)ethylamine TFA salt (0.356 g, 0.392mmol), ammonium formate (0.085 g, 1.35 mmol), and palladium on activatedcharcoal (10% Pd basis, 0.175 g) in MeOH (5 mL) was hydrogenated. After12 h, analysis by LCMS showed the reaction was complete. The crudereaction mixture was filtered over a pad of diatomaceous earth and thefiltrate was concentrated. The crude oil was purified by preparatoryRP-HPLC with a Phenomenex Synergi 10μ Max-RP 80 Å column (150×30 mm)using 10% to 90% MeCN in 0.05% aqueous TFA as the eluent. A total of0.010 g of the title compound was obtained as the TFA salt (0.012 mmol,3%). LCMS RT=1.72 min (Method A); ESI-MS m/z 705.56 [M+H]⁺; 728.33[M+Na]⁺; HRMS m/z 705.4917 [C₃₇H₆₄N₆O₇+H]⁺.

Example 5(2S)-2-(dimethylamino)-N-((2S)-3-hydroxy-1-(((3R,4S,5S)-3-methoxy-1-((2S)-2-((1R,2R)-1-methoxy-2-methyl-3-oxo-3-((2-(piperidin-2-yl)ethyl)amino)propyl)pyrrolidin-1-yl)-5-methyl-1-oxoheptan-4-yl)(methyl)amino)-1-oxopropan-2-yl)-3-methylbutanamide

The title compound was obtained as a byproduct of the hydrogenation stepin Example 3. A total of 0.010 g of the title compound was obtained asthe TFA salt (0.001 mmol). LCMS RT=1.83 min (Method A); ESI-MS m/z697.76 [M+H]⁺; HRMS m/z 697.5226 [C₃₆H₆₈N₆O₇+H]⁺.

Example 6(2S,3R)-2-((S)-2-(dimethylamino)-3-methylbutanamido)-3-hydroxy-N-((3R,4S,5S)-3-methoxy-1-((2S)-2-((1R,2R)-1-methoxy-2-methyl-3-oxo-3-((2-(piperidin-2-yl)ethyl)amino)propyl)pyrrolidin-1-yl)-5-methyl-1-oxoheptan-4-yl)-N-methylbutanamide

The title compound was obtained as a byproduct of the hydrogenation stepin Example 4. A total of 0.010 g of the title compound was obtained asthe TFA salt (0.012 mmol). LCMS RT=1.81 min (Method A); ESI-MS m/z711.43 [M+H]⁺; HRMS m/z 711.5389 [C₃₇H₇₀N₆O₇+H]⁺.

Example 7 (S)-methyl2-((2R,3R)-3-((S)-1-((3R,4S,5S)-4-((2S,3S)-3-azido-2-((S)-2-(dimethylamino)-3-methylbutanamido)-N-methylbutanamido)-3-methoxy-5-methylheptanoyl)pyrrolidin-2-yl)-3-methoxy-2-methylpropanamido)-3-phenylpropanoate

To a stirred room temperature solution of (2S,3S)-Fmoc-Abu(3-N₃)—OH(1.00 g, 2.74 mmol) and H-Dil-OtBu hydrochloride (0.810 g, 2.74 mmol) inEtOAc (10 mL) was added DIEA (0.880 mL, 4.93 mmol). The solution wascooled (0° C.) and stirred for 20 min. DIEA (0.880 mL, 4.93 mmol) wasadded to the reaction mixture. The solution was cooled (0° C.) andstirred for 20 min. CMPI (1.12 g, 4.38 mmol) was added to the reactionmixture and the reaction mixture was allowed to reach room temperature.After 12 h, analysis by LCMS showed the reaction was complete. The crudereaction was washed with 0.1 M HCl (100 mL×2), followed by brine (20mL×2). The organic fraction was dried over a pad of magnesium sulfate,filtered, and concentrated in vacuo to yield crudeFmoc-Abu(3-N₃)-Dil-OtBu (1.12 g, 1.84 mmol) that was used withoutfurther purification.

To a stirred room temperature solution of Fmoc-Abu(3-N₃)-Dil-OtBu (1.00g, 1.65 mmol) in MeCN (10 mL) was added piperidine (2 mL). After 5 h,analysis by LCMS showed the reaction was complete. To the crude reactionmixture was extracted with Hexanes and the MeCN layer was concentratedin vacuo to yield crude H-Abu(3-N₃)-Dil-OtBu that was used withoutfurther purification.

To a stirred room temperature suspension of crude H-Abu(3-N₃)-Dil-OtBuand Dov (0.478 g, 3.29 mmol) in DMF (10 mL) was added DIEA (0.880 mL,4.94 mmol), followed by HATU (1.25 g, 3.29 mmol). After 6 h, analysis byLCMS showed that the reaction was complete. The crude reaction mixturewas diluted with saturated sodium bicarbonate (10 mL) and extracted withEtOAc (40 mL×2). The combined organic fractions were washed with brine,dried over a pad of magnesium sulfate, filtered, and concentrated invacuo. The crude oil was purified by preparatory RP-HPLC with aPhenomenex Synergi 10μ Max-RP 80 Å column (150×30 mm) using 10% to 90%MeCN in 0.05% aqueous TFA as the eluent. A total of 0.670 gDov-Abu(3-N₃)-Dil-OtBu (1.07 mmol, 65%) was obtained as the TFA salt.

To a room temperature solution of Dov-Abu(3-N₃)-Dil-OtBu TFA salt (0.289g, 0.425 mmol) in CH₂Cl₂ (5 mL) was added TFA (5 mL). After 12 h,analysis by LCMS showed the reaction was complete. Volatile organicswere evaporated in vacuo to yield crude Dov-Abu(3-N₃)-Dil-OH TFA saltthat was used without further purification.

To a stirred room temperature suspension of crude Dov-Abu(3-N₃)-Dil-OHTFA salt (1.04 g, 1.82 mmol) and H-Dap-Phe-OMe TFA salt (1.59 g, 3.44mmol) in DMF (10 mL) was added DIEA (1.18 g, 1.60 mL, 9.11 mmol),followed by the addition of HATU (1.74 g, 4.56 mmol). After 10 h,analysis by LCMS showed the reaction was complete. The crude reactionmixture was diluted with saturated sodium bicarbonate (10 mL) andextracted with EtOAc (20 mL×3). The combined organic fractions werewashed with brine, dried over a pad of magnesium sulfate, filtered, andconcentrated in vacuo. The crude oil was purified by preparatory RP-HPLCwith a Phenomenex Gemini NX-C18 10μ 110 Å column (150×30 mm) using 10%to 90% MeCN in 0.1% aqueous formic acid. A total of 935 mg of the titlecompound was obtained as the formic acid salt (1.12 mmol, 49%). LCMSRT=1.09 min (Method A); ESI-MS m/z 787.53 [M+H]⁺; HRMS m/z 787.5072[C₄₀H₆₆N₈O₈+H]⁺.

Example 8 (S)-methyl2-((2R,3R)-3-((S)-1-((3R,4S,5S)-4-((S)-3-amino-2-((S)-2-(dimethylamino)-3-methylbutanamido)-N-methylpropanamido)-3-methoxy-5-methylheptanoyl)pyrrolidin-2-yl)-3-methoxy-2-methylpropanamido)-3-phenylpropanoate

To a stirred room temperature solution of Fmoc-Dpr(Boc)-OH (1.44 g, 3.38mmol) and H-Dil-OtBu hydrochloride (1.00 g, 3.38 mmol) in EtOAc (10 mL)was added DIEA (1.08 mL, 6.08 mmol). The solution was cooled to (0° C.)and stirred for 20 min. Additional DIEA (1.08 mL, 6.08 mmol) was addedto the reaction mixture and the 0° C. solution was stirred for 20 min.CMPI (1.38 g, 5.41 mmol) was then added to the reaction mixture and thereaction mixture was allowed to warm to room temperature. After 12 h,analysis by LCMS showed the reaction was complete. The crude reactionwas washed with 0.1 M HCl (100 mL×2), followed by brine (20 mL×2). Theorganic fraction was dried over a pad of magnesium sulfate, filtered,and concentrated in vacuo. Fmoc-Dpr(Boc)-Dil-OtBu was isolated by flashchromatography on silica gel (silica gel 40 μm, 60 Å, 3.0×17.0 cm) using18% to 90% EtOAc in Hexanes as the eluent. A total of 1.27 g ofFmoc-Dpr(Boc)-Dil-OtBu (1.90 mmol, 56% yield) was obtained.

To a stirred room temperature solution Fmoc-Dpr(Boc)-Dil-OtBu (1.27 g,1.90 mmol) in MeCN (10 mL) was added piperidine (2 mL). After 5 h,analysis by LCMS showed the reaction was complete. The crude reactionmixture was extracted with hexanes and the MeCN layer was concentratedin vacuo to yield crude H-Dpr(Boc)-Dil-OtBu that was used withoutfurther purification.

To a stirred room temperature suspension of crude H-Dpr(Boc)-Dil-OtBuand Dov (0.552 g, 3.80 mmol) in DMF (10 mL) was added DIEA (1.02 mL,5.70 mmol), followed by HATU (1.45 g, 3.80 mmol). After 6 h, analysis byLCMS showed that the reaction was complete. The crude reaction mixturewas diluted with saturated sodium bicarbonate (10 mL) and extracted withEtOAc (40 mL×2). The combined organic fractions were washed with brine,dried over a pad of magnesium sulfate, filtered, and concentrated invacuo. The crude oil was purified by preparatory RP-HPLC with aPhenomenex Synergi 10μ Max-RP 80 Å column (150×30 mm) using 10% to 90%MeCN in 0.05% aqueous TFA as the eluent. A total of 0.598 gDov-Dpr(Boc)-Dil-OtBu (0.871 mmol, 46%) was obtained as the TFA salt.

To a room temperature solution Dov-Dpr(Boc)-Dil-OtBu TFA salt (1.52 g,2.65 mmol) in CH₂Cl₂ (10 mL) was added TFA (5 mL). After 10 h, analysisby LCMS showed the reaction was complete. Volatile organics wereevaporated in vacuo to yield crude Dov-Dpr-Dil-OH TFA salt that was usedwithout further purification.

To a stirred room temperature suspension of Dov-Dpr-Dil-OH TFA salt(1.00 g, 1.89 mmol) and Fmoc-OSu (0.891 g, 2.64 mmol) in CH₂Cl₂ (10 mL)was added DIEA (0.460 mL, 2.64 mmol). After 12 h, analysis by LCMSshowed the reaction was complete. Volatile organics were evaporated invacuo. The crude oil was purified by preparatory RP-HPLC with aPhenomenex Synergi 10μ Max-RP 80 Å column (150×30 mm) using 10% to 90%MeCN in 0.1% aqueous formic acid as the eluent. A total of 1.49 g ofenriched Dov-Dpr(Fmoc)-Dil-OH was obtained as the formic acid salt.

To a stirred 23° C. suspension of Dov-Dpr(Fmoc)-Dil-OH formic acid salt(1.20 g, 1.75 mmol) and H-Dap-Phe-OMe TFA salt (0.982 g, 2.13 mmol) inDMF (10 mL) was added DIEA (0.970 g, 1.30 mL, 7.514 mmol) followed bythe addition of HATU (1.43 g, 3.76 mmol). After 10 h, analysis by LCMSshowed the reaction was complete. The crude reaction was diluted withsaturated sodium bicarbonate (10 mL) and extracted with EtOAc (20 mL×3).The combined organic fractions were washed with brine, dried over a padof magnesium sulfate, filtered, and concentrated in vacuo. The crude oilwas purified by preparatory RP-HPLC with a Phenomenex Gemini NX-C18 10μ110 Å column (150×30 mm) using 10% to 90% MeCN in 0.1% aqueous NH₄OH asthe eluent. A total of 1.01 g of Dov-Dpr(Fmoc)-Dil-Dap-Phe-OMe (1.04mmol, 56%) was obtained.

To a stirred 23° C. solution of Dov-Dpr(Fmoc)-Dil-Dap-Phe-OMe (1.01 g,1.04 mmol) in acetonitrile (10 mL) was added piperidine (5 mL). After 3h, analysis by LCMS showed the reaction was complete. To the crudereaction solution was added hexanes. The acetonitrile layer wasconcentrated in vacuo. The crude oil was purified by preparatory RP-HPLCwith a Phenomenex Gemini NX-C18 10μ 110 Å column (150×30 mm) using 5% to95% MeCN in 0.1% aqueous formic acid as the eluent. A total of 413 g ofthe title compound was obtained as the formic acid salt (0.521 mmol,50%). LCMS RT=2.10 min (Method A); ESI-MS m/z 747.84 [M+H]⁺; HRMS m/z747.5008 [C₃₉H₆₆N₆O₈+H]⁺.

Example 9(S)—N—((S)-3-amino-1-(((3R,4S,5S)-3-methoxy-1-((S)-2-((1R,2R)-1-methoxy-2-methyl-3-oxo-3-((2-(pyridin-2-yl)ethyl)amino)propyl)pyrrolidin-1-yl)-5-methyl-1-oxoheptan-4-yl)(methyl)amino)-1-oxopropan-2-yl)-2-(dimethylamino)-3-methylbutanamide

To a stirred room temperature suspension of crude Dov-Dpr(Fmoc)-Dil-OHand H-Dap-2-(2-pyridyl)ethylamine TFA salt (0.230 g, 0.789 mmol) in DMF(10 mL) was added DIEA (0.511 mL, 2.87 mmol), followed by HATU (0.545 g,1.43 mmol). After 6 h, analysis by LCMS showed the reaction wascomplete. The crude reaction mixture was diluted with saturated sodiumbicarbonate (10 mL) and extracted with EtOAc (25 mL×3). The combinedorganic fractions were washed with brine, dried over a pad of magnesiumsulfate, filtered, and concentrated in vacuo. The crude oil was purifiedby preparatory RP-HPLC with a Phenomenex Synergi 10μ Max-RP 80 Å column(150×30 mm) using 10% to 90% MeCN in 0.05% aqueous TFA as the eluent. Atotal of 0.150 g of Dov-Dpr(Fmoc)-Dil-Dap-2-(2-pyridyl)ethylamine wasobtained as the TFA salt (0.146 mmol, 20%).

To a stirred room temperature solutionDov-Dpr(Fmoc)-Dil-Dap-2-(2-pyridyl)ethylamine TFA salt (0.150 g, 0.146mmol) in MeCN (10 mL) was added piperidine (2 mL). After 5 h, analysisby LCMS showed the reaction was complete. The crude reaction mixture wasextracted with hexanes and the MeCN layer was concentrated in vacuo. Thecrude oil was purified by preparatory RP-HPLC with a Phenomenex Synergi10μ Max-RP 80 Å column (150×30 mm) using 10% to 90% MeCN in 0.05%aqueous TFA as the eluent. A total of 0.089 g of the title compound wasobtained as the TFA salt (0.111 mmol, 75%). LCMS RT=1.52 min (Method A);ESI-MS m/z 690.67 [M+H]⁺; HRMS m/z 690.4917 [C₃₆H₆₃N₇O₆+H]⁺.

Example 10 (S)-methyl2-((2R,3R)-3-((S)-1-((3R,4S,5S)-4-((S)-3-azido-2-((S)-2-(dimethylamino)-3-methylbutanamido)-N-methylpropanamido)-3-methoxy-5-methylheptanoyl)pyrrolidin-2-yl)-3-methoxy-2-methylpropanamido)-3-phenylpropanoate

To a stirred room temperature solution of N-Boc-4-Azido-Alaninedicyclohexylamine salt (1.02 g, 2.47 mmol) and H-Dil-OtBu hydrochloride(0.730 g, 2.74 mmol) in EtOAc (10 mL) was added DIEA (0.792 mL, 4.44mmol). The solution was cooled (0° C.) and stirred for 20 min. DIEA(0.792 mL, 4.44 mmol) was added to the reaction mixture. The solutionwas cooled (0° C.) and stirred for 20 min. CMPI (1.01 g, 3.95 mmol) wasadded to the reaction mixture and the reaction mixture was allowed toreach room temperature. After 12 h, analysis by LCMS showed the reactionwas complete. The crude reaction was washed with 0.1 M HCl (100 mL×2),followed by brine (20 mL×2). The organic fraction was dried over a padof magnesium sulfate, filtered, and concentrated in vacuo to yield crudeN-Boc-4-Azido-Ala-Dil-OtBu (1.08 g, 2.29 mmol) that was used withoutfurther purification.

To a room temperature solution of N-Boc-4-Azido-Ala-Dil-OtBu (1.08 g,2.29 mmol) in CH₂Cl₂ (5 mL) was added TFA (3 mL). After 10 h, analysisby LCMS showed the reaction was complete. Volatile organics wereevaporated in vacuo to yield crude H-4-Azido-Ala-Dil-OH TFA salt thatwas used without further purification.

To a stirred room temperature suspension of Dov (0.739 g, 5.09 mmol) inDMF (10 mL) was added DIEA (1.36 mL, 7.63 mmol), followed by HATU (1.95g, 5.09 mmol). After 10 min crude H-4-Azido-Ala-Dil-OH was added to thereaction mixture. After 6 h, analysis by LCMS showed that the reactionwas complete. The crude oil was purified by preparatory RP-HPLC with aPhenomenex Synergi 10μ Max-RP 80 Å column (150×30 mm) using 10% to 90%MeCN in 0.05% aqueous TFA as the eluent. A total of 0.130 gDov-4-Azido-Ala-Dil-OH (0.261 mmol, 10%) was obtained as the TFA salt.

To a stirred room temperature suspension of Dov-4-Azido-Ala-Dil-OH TFAsalt (0.130 g, 0.261) and H-Dap-Phe-OMe TFA salt (0.205 g, 0.588 mmol)in DMF (10 mL) was added DIEA (0.140 mL, 0.783 mmol), followed by HATU(0.198 g, 0.522 mmol). After 6 h, analysis by LCMS showed the reactionwas complete. The crude reaction mixture was diluted with saturatedsodium bicarbonate (10 mL) and extracted with EtOAc (40 mL×2). Thecombined organic fractions were washed with brine, dried over a pad ofmagnesium sulfate, filtered, and concentrated in vacuo. The crude oilwas purified by preparatory RP-HPLC with a Phenomenex Synergi 10μ Max-RP80 Å column (150×30 mm) using 10% to 90% MeCN in 0.05% aqueous TFA asthe eluent. A total of 0.015 g of the title compound was obtained as theTFA salt (0.017 mmol, 7%). LCMS RT=2.36 min (Method A); ESI-MS m/z773.48 [M+H]⁺; HRMS m/z 773.4916 [C₃₉H₆₄N₈O₈+H]⁺.

Example 11 (S)-methyl2-((2R,3R)-3-((S)-1-((3R,4S,5S)-4-((S)-4-azido-2-((S)-2-(dimethylamino)-3-methylbutanamido)-N-methylbutanamido)-3-methoxy-5-methylheptanoyl)pyrrolidin-2-yl)-3-methoxy-2-methylpropanamido)-3-phenylpropanoate

To a stirred room temperature solution of N-Boc-4-Azido-homoalaninedicyclohexylamine salt (1.01 g, 2.37 mmol) and H-Dil-OtBu hydrochloride(0.700 g, 2.37 mmol) in EtOAc (10 mL) was added DIEA (0.759 mL, 4.26mmol). The solution was cooled (0° C.) and stirred for 20 min. DIEA(0.759 mL, 4.26 mmol) was added to the reaction mixture. The solutionwas cooled (0° C.) and stirred for 20 min. CMPI (0.967 g, 3.79 mmol) wasadded to the reaction mixture and the reaction mixture was allowed toreach room temperature. After 12 h, analysis by LCMS showed the reactionwas complete. The crude reaction was washed with 0.1 M HCl (100 mL×2),followed by brine (20 mL×2). The organic fraction was dried over a padof magnesium sulfate, filtered, and concentrated in vacuo to yield crudeN-Boc-4-Azido-homoAla-Dil-OtBu (1.09 g, 2.25 mmol) that was used withoutfurther purification.

To a room temperature solution of N-Boc-4-Azido-homoAla-Dil-OtBu (1.09g, 2.25 mmol) in CH₂Cl₂ (5 mL) was added TFA (3 mL). After 10 h,analysis by LCMS showed the reaction was complete. Volatile organicswere evaporated in vacuo to yield crude H-4-Azido-homoAla-Dil-OH TFAsalt that was used without further purification.

To a stirred room temperature suspension of Dov (0.652 g, 4.49 mmol) inDMF (10 mL) was added DIEA (1.20 mL, 6.73 mmol), followed by HATU (1.71g, 4.49 mmol). After 10 min crude H-4-Azido-homoAla-Dil-OH TFA salt wasadded to the reaction mixture. After 6 h, analysis by LCMS showed thatthe reaction was complete. The crude oil was purified by preparatoryRP-HPLC with a Phenomenex Synergi 10μ Max-RP 80 Å column (150×30 mm)using 10% to 90% MeCN in 0.05% aqueous TFA as the eluent. A total of0.127 g Dov-4-Azido-homoAla-Dil-OH (0.223 mmol, 10%) was obtained as theTFA salt.

To a stirred room temperature suspension of Dov-4-Azido-homoAla-Dil-OHTFA salt (0.127 g, 0.223) and H-Dap-Phe-OMe TFA salt (0.198 g, 0.568mmol) in DMF (10 mL) was added DIEA (0.140 mL, 0.783 mmol), followed byHATU (0.198 g, 0.522 mmol). After 6 h, analysis by LCMS showed thereaction was complete. The crude reaction mixture was diluted withsaturated sodium bicarbonate (10 mL) and extracted with EtOAc (40 mL×2).The combined organic fractions were washed with brine, dried over a padof magnesium sulfate, filtered, and concentrated in vacuo. The crude oilwas purified by preparatory RP-HPLC with a Phenomenex Synergi 10μ Max-RP80 Å column (150×30 mm) using 10% to 90% MeCN in 0.05% aqueous TFA asthe eluent. A total of 0.015 g of the title compound was obtained as theTFA salt (0.017 mmol, 7%). LCMS RT=2.38 min (Method A); ESI-MS m/z787.49 [M+H]⁺; HRMS m/z 787.5078 [C₄₀H₆₆N₈O₈+H]⁺.

Example 12 (S)-methyl2-((2R,3R)-3-((S)-1-((3R,4S,5S)-4-((S)-4-amino-2-((S)-2-(dimethylamino)-3-methylbutanamido)-N-methylbutanamido)-3-methoxy-5-methylheptanoyl)pyrrolidin-2-yl)-3-methoxy-2-methylpropanamido)-3-phenylpropanoate

To a stirred room temperature solution of Fmoc-Dab(Boc)-OH (1.54 g, 3.38mmol) and H-Dil-OtBu hydrochloride (1.00 g, 3.38 mmol) in EtOAc (10 mL)was added DIEA (1.08 mL, 6.08 mmol). The solution was cooled (0° C.) andstirred for 20 min. Additional DIEA (1.08 mL, 6.08 mmol) was added tothe reaction mixture, and the 0° C. solution was stirred for 20 min.Then CMPI (1.38 g, 5.41 mmol) was added to the reaction mixture and thereaction mixture was allowed to warm to room temperature. After 12 h,analysis by LCMS showed the reaction was complete. The crude reactionwas washed with 0.1 M HCl (100 mL×2), followed by brine (20 mL×2). Theorganic fraction was dried over a pad of magnesium sulfate, filtered,and concentrated in vacuo. Fmoc-Dab(Boc)-Dil-OtBu was isolated by flashchromatography on silica gel (silica gel 40 μm, 60 Å, 3.0×17.0 cm) using18% to 90% EtOAc in hexanes as the eluent. A total of 1.18 g ofFmoc-Dpr(Boc)-Dil-OtBu (1.73 mmol, 51% yield) was obtained.

To a stirred room temperature solution Fmoc-Dab(Boc)-Dil-OtBu (1.18 g,1.73 mmol) in MeCN (10 mL) was added piperidine (2 mL). After 5 h,analysis by LCMS showed the reaction was complete. The crude reactionmixture was extracted with Hexanes and the MeCN layer was concentratedin vacuo to yield crude H-Dab(Boc)-Dil-OtBu that was used withoutfurther purification.

To a stirred room temperature suspension of crude H-Dab(Boc)-Dil-OtBuand Dov (0.502 g, 3.46 mmol) in DMF (10 mL) was added DIEA (0.925 mL,5.19 mmol), followed by HATU (1.32 g, 3.46 mmol). After 6 h, analysis byLCMS showed that the reaction was complete. The crude reaction mixturewas diluted with saturated sodium bicarbonate (10 mL) and extracted withEtOAc (40 mL×2). The combined organic fractions were washed with brine,dried over a pad of magnesium sulfate, filtered, and concentrated invacuo. The crude oil was purified by preparatory RP-HPLC with aPhenomenex Synergi 10μ Max-RP 80 Å column (150×30 mm) using 10% to 90%MeCN in 0.05% aqueous TFA as the eluent. A total of 0.410 g ofDov-Dab(Boc)-Dil-OtBu (0.583 mmol, 34%) was obtained as the TFA salt.

To a room temperature solution Dov-Dab(Boc)-Dil-OtBu TFA salt (0.240 g,0.419 mmol) in CH₂Cl₂ (5 mL) was added TFA (2 mL). After 10 h, analysisby LCMS showed the reaction was complete. Volatile organics wereevaporated in vacuo to yield crude Dov-Dab-Dil-OH TFA salt that was usedwithout further purification.

To a stirred room temperature suspension of Dov-Dab-Dil-OH TFA salt(0.175 g, 0.406 mmol) and Fmoc-OSu (0.151 g, 0.447 mmol) in CH₂Cl₂ (5mL) was added DIEA (0.080 mL, 0.447 mmol). After 12 h, analysis by LCMSshowed the reaction was complete. Volatile organics were evaporated invacuo. The crude reaction mixture was dissolved in EtOAc and was washedwith 0.1 M HCl (100 mL×2), followed by brine (20 mL×2). The organicfraction was dried over a pad of magnesium sulfate, filtered, andconcentrated in vacuo to yield crude Dov-Dab(Fmoc)-Dil-OH that was usedwithout further purification.

To a stirred room temperature suspension of crude Dov-Dab(Fmoc)-Dil-OH(0.363 g, 0.556 mmol) and H-Dap-Phe-OMe TFA salt (0.194 g, 0.420 mmol)in DMF (10 mL) was added DIEA (0.291 mL, 1.67 mmol), followed by HATU(0.424 g, 1.11 mmol). After 10 h, analysis by LCMS showed the reactionwas complete. The crude reaction mixture was diluted with saturatedsodium bicarbonate (10 mL) and extracted with EtOAc (30 mL×2). Thecombined organic fractions were washed with brine, dried over a pad ofmagnesium sulfate, filtered, and concentrated in vacuo. The crude oilwas purified by preparatory RP-HPLC with a Phenomenex Synergi 10μ Max-RP80 Å column (150×30 mm) using 10% to 90% MeCN in 0.2% aqueous formicacid as the eluent. A total of 449 mg of enrichedDov-Dab(Fmoc)-Dil-Dap-Phe-OMe was obtained as the formic acid salt.

To a stirred room temperature solution enrichedDov-Dab(Fmoc)-Dil-Dap-Phe-OMe formic acid salt (0.449 g) in MeCN (10 mL)was added piperidine (5 mL). After 10 h, analysis by LCMS showed thereaction was complete. The crude reaction mixture was extracted withhexanes and the MeCN layer was concentrated in vacuo. The crude oil waspurified by RP-HPLC with a Phenomenex Gemini NX C18 10μ Max-RP 110 Åcolumn (150×30 mm) using 10% to 90% MeCN in 0.1% aqueous formic acid asthe eluent. A total of 43.0 g of the title compound was obtained as theformic acid salt (0.053 mmol, 13%). LCMS RT=1.24 min (Method B); ESI-MSm/z 761.57 [M+H]⁺; HRMS m/z 761.5165 [C₄₀H₆₈N₆O₈+H]⁺.

Example 13(S)-2-((S)-2-(aminooxy)-3-methylbutanamido)-N-((3R,4S,5S)-3-methoxy-1-((S)-2-((1R,2R)-1-methoxy-2-methyl-3-oxo-3-((2-(pyridin-2-yl)ethyl)amino)propyl)pyrrolidin-1-yl)-5-methyl-1-oxoheptan-4-yl)-N,3-dimethylbutanamide

To a solution of H-Dil-OtBu hydrochloride (0.60 g, 2.03 mmol) andFmoc-Val-OH (0.829 g, 2.44 mmol) stirring in EtOAc (3 mL) was added DIEA(0.65 mL, 3.7 mmol). The reaction was cooled to 0° C. and stirred for 20min, followed by addition of DIEA (0.65 mL, 3.7 mmol). The reactionmixture was cooled (0° C.) for another 20 min, followed by the additionof CMPI (0.83 g, 3.7 mmol). After 8 h, analysis by LCMS showed thereaction was complete. The reaction mixture was washed with 1 M HCl (25mL×2) and brine (50 mL). The organic phase was dried over magnesiumsulfate, filtered, and concentrated in vacuo. Fmoc-Val-Dil-OtBu wasisolated by flash chromatography on silica gel (silica gel 40 μm, 60 Å,3.0×17.0 cm) using 18% to 90% EtOAc in hexanes as the eluent. A total of1.1 g of Fmoc-Val-Dil-OtBu (1.9 mmol, 93% yield) was obtained.

To a stirred room temperature suspension of Fmoc-Val-Dil-OtBu (0.883 g,1.52 mmol) and Boc-Dap-2-(2-pyridyl)ethylamine (0.451 g, 1.52 mmol) inCH₂Cl₂ (10 mL) was added TFA (5 mL). After 8 h, analysis by LCMS showedthe reaction was complete. Volatile organics were evaporated in vacuo toyield crude Fmoc-Val-Dil-OH and H-Dap-2-(2-pyridyl)ethylamine TFA salt,which were used without further purification.

To a stirred room temperature suspension of crudeH-Dap-2-(2-pyridyl)ethylamine TFA salt and Fmoc-Val-Dil-OH in EtOAc (2mL) was added DIEA (1.10 mL, 6.08 mmol), followed by DEPC (0.92 mL, 6.08mmol). After 15 h, analysis by LCMS showed the reaction was complete.The reaction was washed with a saturated NaHCO₃ solution (50 mL)followed by water (50 mL×2). The organic fraction was filtered through apad of magnesium sulfate and concentrated in vacuo. The resultingviscous oil was purified by flash chromatography on silica gel (silicagel 40 μm, 60 Å, 23×123 mm) using 5% to 10% MeOH in CH₂Cl₁₂ as theeluent. A total of 0.888 g of Fmoc-Val-Dil-Dap-2-(2-pyridyl)ethylamine(1.11 mmol, 73% yield) was obtained.

To a stirred room temperature solution ofFmoc-Val-Dil-Dap-2-(2-pyridyl)ethylamine (1.75 g, 2.19 mmol) in CH₂Cl₂(5 mL) was added piperidine (5 mL). After 8 h, analysis by LCMS showedthe reaction was complete. Volatile organics were evaporated in vacuo toyield crude H-Val-Dil-Dap-2-(2-pyridyl)ethylamine that was used withoutfurther purification.

To a stirred room temperature suspension of crudeH-Val-Dil-Dap-2-(2-pyridyl)ethylamine and N-Boc-N-hydroxyVal-OH (0.390g, 1.67 mmol) in DMF (5 mL) was added DIEA (0.797 mL, 5.02 mmol),followed by HATU (1.28 g, 3.34 mmol). After 8 h, analysis by LCMS showedthat the reaction was complete. The crude reaction mixture was dilutedwith saturated sodium bicarbonate (10 mL) and extracted with EtOAc (50mL×3). The combined organic fractions were washed with brine, dried overa pad of magnesium sulfate, filtered, and concentrated in vacuo to yield0.563 g of crude N-Boc-N-hydroxyVal-Val-Dil-Dap-2-(2-pyridyl)ethylamine(0.712 mmol, 43%) that was used without further purification.

To a room temperature solution ofN-Boc-N-hydroxyVal-Val-Dil-Dap-2-(2-pyridyl)ethylamine (0.563 g, 0.712mmol) in DMF (2 mL) was added TFA (2 mL). After 2 h, analysis by LCMSshowed the reaction was complete. Volatile organics were evaporated invacuo. The crude oil was purified by preparatory RP-HPLC with aPhenomenex Synergi 10μ Max-RP 80 Å column (150×30 mm) using 10% to 90%MeCN in 0.05% aqueous TFA as the eluent. A total of 0.209 g of the titlecompound was obtained as the TFA salt (0.302 mmol, 43%). LCMS RT=1.70min (Method A); ESI-MS m/z 691.53 [M+H]⁺; HRMS m/z 691.4755[C₃₆H₆₂N₆O₇+H]⁺.

Example 14((2R,3R)-3-((S)-1-((3R,4S,5S)-4-((S)-2-((S)-2-(dimethylamino)-3-hydroxypropanamido)-N,3-dimethylbutanamido)-3-methoxy-5-methylheptanoyl)pyrrolidin-2-yl)-3-methoxy-2-methylpropanoyl)-L-phenylalanine

To a stirred room temperature solution of H-Ser(Bzl)-OH (0.500 g, 2.56mmol) and paraformaldehyde (1.15 g, 38.4 mmol) in MeOH (10 mL) was addedHCO₂NH₄ (0.808 g, 12.8 mmol) and palladium on activated charcoal (10% Pdbasis, 0.250 g). After 72 h, analysis by LCMS showed the reaction wascomplete. The crude reaction mixture was filtered over a pad ofdiatomaceous earth and the filtrate was concentrated. The crude oil waspurified by preparatory RP-HPLC with a Phenomenex Synergi 10μ Max-RP 80Å column (150×30 mm) using 10% to 90% MeCN in 0.05% aqueous TFA as theeluent. A total of 0.168 g of N,N-dimethylSer(Bzl)-OH (0.498 mmol, 19%)was obtained as the TFA salt.

To a stirred room temperature solution of crude Fmoc-Val-Dil-OtBu (13.3g, 22.8 mmol) in CH₂Cl₂ (20 mL) was added piperidine (15 mL). After 8 h,analysis by LCMS showed the reaction was complete. Volatile organicswere evaporated in vacuo to yield crude H-Val-Dil-OtBu that was usedwithout further purification.

To a stirred room temperature suspension of crude H-Val-Dil-OtBu andN,N-dimethylSer(Bzl)-OH TFA salt (0.780 g, 3.49 mmol) in DMF (10 mL) wasadded DIEA (1.25 mL, 6.99 mmol), followed by HATU (1.77 g, 4.66 mmol).After 6 h, analysis by LCMS showed that the reaction was complete. Thecrude reaction mixture was diluted with saturated sodium bicarbonate (10mL) and extracted with EtOAc (40 mL×2). The combined organic fractionswere washed with brine, dried over a pad of magnesium sulfate, filtered,and concentrated in vacuo. The crude oil was purified by preparatoryRP-HPLC with a Phenomenex Synergi 10μ Max-RP 80 Å column (150×30 mm)using 10% to 90% MeCN in 0.05% aqueous TFA as the eluent. A total of0.220 g of N,N-dimethylSer(Bzl)-Val-Dil-OtBu (0.325 mmol, 14%) wasobtained as the TFA salt.

To a room temperature solution N,N-dimethylSer(Bzl)-Val-Dil-OtBu TFAsalt (0.220 g, 0.325 mmol) in CH₂Cl₂ (5 mL) was added TFA (2 mL). After10 h, analysis by LCMS showed the reaction was complete. Volatileorganics were evaporated in vacuo to yield crudeN,N-dimethylSer(Bzl)-Val-Dil-OH TFA salt that was used without furtherpurification.

To a stirred room temperature suspension of crudeN,N-dimethylSer(Bzl)-Val-Dil-OH TFA salt and H-Dap-Phe-OMe TFA salt(0.166 g, 0.477 mmol) in DMF (5 mL) was added DIEA (0.207 mL, 1.30mmol), followed by HATU (0.330 g, 0.868 mmol). After 4 h, analysis byLCMS showed the reaction was complete. The crude reaction mixture wasdiluted with saturated sodium bicarbonate (10 mL) and extracted withEtOAc (40 mL×2). The combined organic fractions were washed with brine,dried over a pad of magnesium sulfate, filtered, and concentrated invacuo. The crude oil was purified by preparatory RP-HPLC with aPhenomenex Synergi 10μ Max-RP 80 Å column (150×30 mm) using 10% to 90%MeCN in 0.05% aqueous TFA as the eluent. A total of 0.201 gN,N-dimethylSer(Bzl)-Val-Dil-Dap-Phe-OMe (0.211 mmol, 49%) was obtainedas the TFA salt.

To a stirred room temperature suspension ofN,N-dimethylSer(Bzl)-Val-Dil-Dap-Phe-OMe TFA salt (0.201 g, 0.211 mmol)and NH₄HCO₂ (2.00 g, 31.7 mmol) in MeOH (6 mL) and water (1.0 mL) wasadded 10% Pd/C (10 mg). The solution was stirred vigorously at roomtemperature. After 10 h, analysis by LCMS showed the reaction wascomplete, along with hydrolysis of the methyl ester of phenylalanine.The reaction mixture was filtered through a pad of a diatomaceous earthand the filtrate was concentrated. The crude oil was purified bypreparatory RP-HPLC with a Phenomenex Synergi 10μ Max-RP 80 Å column(150×30 mm) using 10% to 90% MeCN in 0.05% aqueous TFA as the eluent. Atotal of 63.0 mg of the title compound was obtained as the TFA salt(0.074 mmol, 31%). LCMS RT=2.01 min (Method A); ESI-MS m/z 734.61[M+H]⁺; HRMS m/z 734.4715 [C₃₈H₆₃N₅O₉+H]⁺.

Example 15((2R,3R)-3-((S)-1-((3R,4S,5S)-4-((S)-2-((2S,3R)-2-(dimethylamino)-3-hydroxybutanamido)-N,3-dimethylbutanamido)-3-methoxy-5-methylheptanoyl)pyrrolidin-2-yl)-3-methoxy-2-methylpropanoyl)-L-phenylalanine

To a stirred room temperature solution of H-Thr(Bzl)-OH (2.00 g, 9.56mmol) and paraformaldehyde (9.87 g, 95.6 mmol) in MeOH (40 mL) was addedHCO₂NH₄ (3.01 g, 47.8 mmol) and palladium on activated charcoal (10% Pdbasis, 1.00 g). After 72 h, analysis by LCMS showed the reaction wascomplete. The crude reaction mixture was filtered over a pad ofdiatomaceous earth and the filtrate was concentrated. The crude oil waspurified by preparatory RP-HPLC with a Phenomenex Synergi 10μ Max-RP 80Å column (150×30 mm) using 10% to 90% MeCN in 0.05% aqueous TFA as theeluent. A total of 1.54 g of N,N-dimethylThr(Bzl)-OH (4.38 mmol, 19%)was obtained as the TFA salt.

To a stirred room temperature suspension of crude H-Val-Dil-OtBu andN,N-dimethylThr(Bzl)-OH TFA salt (1.21 g, 5.10 mmol) in DMF (20 mL) wasadded DIEA (1.82 mL, 10.2 mmol), followed by HATU (2.59 g, 6.80 mmol).After 6 h, analysis by LCMS showed that the reaction was complete. Thecrude reaction mixture was diluted with saturated sodium bicarbonate (15mL) and extracted with EtOAc (40 mL×2). The combined organic fractionswere washed with brine, dried over a pad of magnesium sulfate, filtered,and concentrated in vacuo. The crude oil was purified by preparatoryRP-HPLC with a Phenomenex Synergi 10μ Max-RP 80 Å column (150×30 mm)using 10% to 90% MeCN in 0.05% aqueous TFA as the eluent. A total of0.161 g of N,N-dimethylThr(Bzl)-Val-Dil-OtBu (0.234 mmol, 7%) wasobtained as the TFA salt.

To a room temperature solution N,N-dimethylThr(Bzl)-Val-Dil-OtBu TFAsalt (0.220 g, 0.325 mmol) in CH₂Cl₂ (5 mL) was added TFA (2 mL). After10 h, analysis by LCMS showed the reaction was complete. Volatileorganics were evaporated in vacuo to yield crudeN,N-dimethylThr(Bzl)-Val-Dil-OH TFA salt that was used without furtherpurification.

To a stirred room temperature suspension of crudeN,N-dimethylThr(Bzl)-Val-Dil-OH TFA salt and H-Dap-Phe-OMe TFA salt(0.118 g, 0.339 mmol) in DMF (5 mL) was added DIEA (0.147 mL, 0.927mmol), followed by HATU (0.235 g, 0.618 mmol). After 4 h, analysis byLCMS showed the reaction was complete. The crude reaction mixture wasdiluted with saturated sodium bicarbonate (10 mL) and extracted withEtOAc (40 mL×2). The combined organic fractions were washed with brine,dried over a pad of magnesium sulfate, filtered, and concentrated invacuo. The crude oil was purified by preparatory RP-HPLC with aPhenomenex Synergi 10μ Max-RP 80 Å column (150×30 mm) using 10% to 90%MeCN in 0.05% aqueous TFA as the eluent. A total of 0.186 gN,N-dimethylThr(Bzl)-Val-Dil-Dap-Phe-OMe (0.193 mmol, 62%) was obtainedas the TFA salt.

To a stirred room temperature suspension ofN,N-dimethylThr(Bzl)-Val-Dil-Dap-Phe-OMe TFA salt (0.186 g, 0.193 mmol)and NH₄HCO₂ (2.00 g, 31.7 mmol) in MeOH (6 mL) and water (1.0 mL) wasadded 10% Pd/C (100 mg). The solution was stirred vigorously at roomtemperature. After 72 h, analysis by LCMS showed the reaction wascomplete, along with hydrolysis of the methyl ester of phenylalanine.The reaction mixture was filtered through a pad of diatomaceous earthand the filtrate was concentrated. The crude oil was purified bypreparatory RP-HPLC with a Phenomenex Synergi 10μ Max-RP 80 Å column(150×30 mm) using 10% to 90% MeCN in 0.05% aqueous TFA as the eluent. Atotal of 63.0 mg of the title compound was obtained as the TFA salt(0.073 mmol, 38%). LCMS RT=2.04 min (Method A); ESI-MS m/z 748.58[M+H]⁺; HRMS m/z 748.4854 [C₃₉H₆₅N₅O₉+H]⁺.

Example 16(S)-2-((2R,3R)-3-((S)-1-((3R,4S,5S)-4-((2S,3S)-3-azido-2-((S)-2-(dimethylamino)-3-methylbutanamido)-N-methylbutanamido)-3-methoxy-5-methylheptanoyl)pyrrolidin-2-yl)-3-methoxy-2-methylpropanamido)-3-phenylpropanoicAcid

To a stirred room temperature solution of Dov-Abu(3-N₃)-Dil-Dap-Phe-OMeTFA salt (60 mg, 0.067 mmol) in MeOH (0.1 mL) and THF (0.1 mL) was addedlithium hydroxide monohydrate (9.6 mg, 0.229 mmol) in water (0.1 mL).After 12 h, analysis by LCMS showed the reaction was complete. The crudeoil was purified by preparatory RP-HPLC with a Phenomenex Synergi 10μMax-RP 80 Å column (150×30 mm) using 10% to 90% MeCN in 0.05% aqueousTFA as the eluent. A total of 55.0 mg of the title compound was obtainedas the TFA salt (0.062 mmol, 93%). LCMS RT=2.25 min (Method A); ESI-MSm/z 773.45 [M+H]⁺; HRMS m/z 773.49119 [C₃₉H₆₄N₈O₈+H]⁺.

Example 17(S)—N-((3R,4S,5S)-1-((S)-2-((1R,2R)-3-(((S)-1-amino-1-oxo-3-phenylpropan-2-yl)amino)-1-methoxy-2-methyl-3-oxopropyl)pyrrolidin-1-yl)-3-methoxy-5-methyl-1-oxoheptan-4-yl)-2-((2S,3R)-2-(dimethylamino)-3-hydroxybutanamido)-N,3-dimethylbutanamide

To a stirred room temperature solution ofN,N-dimethylThr-Val-Dil-Dap-Phe-OH TFA salt (40 mg, 0.053 mmol) inCH₂Cl₂ (10 mL) was added ammonium chloride (5.72 mg, 0.107 mmol), DIEA(28.6 μL, 0.160 mmol) and EDCI (30.3 mg, 0.107 mmol). After 12 h,analysis by LCMS showed the reaction was complete. The crude oil waspurified by preparatory RP-HPLC with a Phenomenex Synergi 10μ Max-RP 80Å column (150×30 mm) using 10% to 90% MeCN in 0.05% aqueous TFA as theeluent. A total of 28.0 mg of the title compound was obtained as the TFAsalt (0.037 mmol, 70%). LCMS RT=2.14 min (Method A); ESI-MS m/z 747.61[M+H]⁺; HRMS m/z 747.5017 [C₃₉H₆₆N₆O₈+H]⁺.

Example 18(S)—N-((3R,4S,5S)-1-((S)-2-((1R,2R)-3-(((S)-1-amino-1-oxo-3-phenylpropan-2-yl)amino)-1-methoxy-2-methyl-3-oxopropyl)pyrrolidin-1-yl)-3-methoxy-5-methyl-1-oxoheptan-4-yl)-2-((S)-2-(dimethylamino)-3-hydroxypropanamido)-N,3-dimethylbutanamide

To a stirred room temperature solution ofN,N-dimethylSer-Val-Dil-Dap-Phe-OH TFA salt (12 mg, 0.016 mmol) inCH₂Cl₂ (10 mL) was added ammonium chloride (1.00 mg, 0.019 mmol), DIEA(3.21 μL, 0.018 mmol) and EDCI (5.09 mg, 0.018 mmol). After 12 h,analysis by LCMS showed the reaction was complete. The crude oil waspurified by preparatory RP-HPLC with a Phenomenex Synergi 10μ Max-RP 80Å column (150×30 mm) using 10% to 90% MeCN in 0.05% aqueous TFA as theeluent. A total of 10.0 mg of the title compound was obtained as the TFAsalt (0.014 mmol, 83%). LCMS RT=2.07 min (Method A); ESI-MS m/z 733.63[M+H]⁺; HRMS m/z 733.4866 [C₃₈H₆₄N₆O₈+H]⁺.

Example 19 (S)-methyl2-((2R,3R)-3-((S)-1-((3R,4S,5S)-4-((R)-2-((S)-2-(dimethylamino)-3-methylbutanamido)-3-mercapto-N-methylpropanamido)-3-methoxy-5-methylheptanoyl)pyrrolidin-2-yl)-3-methoxy-2-methylpropanamido)-3-phenylpropanoate

To a stirred room temperature solution of Fmoc-Cys(Trt)-OH (3.96 g, 6.76mmol) and H-Dil-OtBu hydrochloride (2.00 g, 6.76 mmol) in EtOAc (15 mL)was added DIEA (2.17 mL, 12.2 mmol). The solution was cooled (0° C.) andstirred for 20 min. Additional DIEA (2.17 mL, 12.2 mmol) was added tothe reaction mixture, and the 0° C. solution was stirred for 20 min.Then CMPI (2.76 g, 10.8 mmol) was added to the reaction mixture and thereaction mixture was allowed to reach room temperature. After 12 h,analysis by LCMS showed the reaction was complete. The crude reactionwas washed with 0.1 M HCl (100 mL×2), followed by brine (20 mL×2). Theorganic fraction was dried over a pad of magnesium sulfate, filtered andconcentrated in vacuo. Fmoc-Cys(Trt)-Dil-OtBu was isolated by flashchromatography on silica gel (silica gel 40 μm, 60 Å, 3.0×17.0 cm) using18% to 90% EtOAc in hexanes as the eluent. A total of 4.73 g ofFmoc-Cys(Trt)-Dil-OtBu (5.72 mmol, 85% yield) was obtained.

To a stirred room temperature solution of Fmoc-Cys(Trt)-Dil-OtBu (2.61g, 3.16 mmol) in MeCN (10 mL) was added piperidine (5 mL). After 5 h,analysis by LCMS showed the reaction was complete. The crude reactionmixture was extracted with hexanes and the MeCN layer was concentratedin vacuo to yield crude H-Cys(Trt)-Dil-OtBu that was used withoutfurther purification.

To a stirred room temperature suspension of crude H-Cys(Trt)-Dil-OtBuand Dov (0.916 g, 6.31 mmol) in DMF (15 mL) was added DIEA (1.69 mL,9.47 mmol), followed by HATU (2.40 g, 6.31 mmol). After 8 h, analysis byLCMS showed that the reaction was complete. The crude reaction mixturewas diluted with saturated sodium bicarbonate (10 mL) and extracted withEtOAc (40 mL×2). The combined organic fractions were washed with brine,dried over a pad of magnesium sulfate, filtered, and concentrated invacuo. The crude oil was purified by preparatory RP-HPLC with aPhenomenex Synergi 10μ Max-RP 80 Å column (150×30 mm) using 10% to 90%MeCN in 0.05% aqueous TFA as the eluent. A total of 1.40 gDov-Cys(Trt)-Dil-OtBu (1.66 mmol, 52%) was obtained as the TFA salt.

To a room temperature solution of Dov-Cys(Trt)-Dil-OtBu TFA salt (1.40g, 1.66 mmol) in CH₂Cl₂ (10 mL) was added TFA (5 mL). After 10 h,analysis by LCMS showed the reaction was complete. Volatile organicswere evaporated in vacuo to yield crude Dov-Cys(Trt)-Dil-OH TFA saltthat was used without further purification.

To a stirred room temperature suspension of Dov-Cys(Trt)-Dil-OH TFA salt(0.225 g, 285 mmol) and H-Dap-Phe-OMe TFA salt (0.139 g, 0.301 mmol) inDMF (10 mL) was added DIEA (0.237 mL, 1.33 mmol), followed by HOBt(0.102 g, 0.666 mmol) and HATU (0.253 g, 0.666 mmol). After overnightstirring, analysis by LCMS showed the reaction was complete. The crudereaction mixture was diluted with saturated sodium bicarbonate (10 mL)and extracted with EtOAc (50 mL×3). The combined organic fractions werewashed with brine, dried over a pad of magnesium sulfate, filtered, andconcentrated in vacuo and used without further purification.

To a room temperature solution of Dov-Cys(Trt)-Dil-Dap-Phe-OMe TFA salt(0.568 g, 0.507 mmol) was added TFA (10 mL). After 12 h at 60° C.,analysis by LCMS showed the reaction was complete. Volatile organicswere evaporated in vacuo. The crude oil was purified by preparatoryRP-HPLC with a Phenomenex Synergi 10μ Max-RP 80 Å column (150×30 mm)using 10% to 90% MeCN in 0.05% aqueous TFA as the eluent. A total of10.0 mg of the title compound was obtained as the disulfide bridge dimerTFA salt (0.011 mmol, 3%). LCMS RT=2.50 min (Method A); ESI-MS m/z764.60 [M+H]⁺; HRMS m/z 763.4547 z=2.

Example 20(S)-2-((2R,3R)-3-((S)-1-((3R,4S,5S)-4-((R)-2-((S)-2-(dimethylamino)-3-methylbutanamido)-3-mercapto-N-methylpropanamido)-3-methoxy-5-methylheptanoyl)pyrrolidin-2-yl)-3-methoxy-2-methylpropanamido)-3-phenylpropanoicAcid

To a stirred room temperature solution of Boc-Dap-OH dicyclohexylaminesalt (6.47 g, 13.8 mmol) and H-Phe-OtBu HCl salt (3.91 g, 15.2 mmol) inDCM (20 mL) was added DIEA (8.78 mL, 55.2 mmol), followed by DEPC (3.12mL, 20.7 mmol). After 8 h, analysis by LCMS showed the reaction wascomplete. The volatile organic were evaporated in vacuo to give crudeproduct that was used without further purification.

To a stirred room temperature solution of Boc-Dap-Phe-OtBu (5.25 g, 10.7mmol) in CH₂Cl₂ (10 mL) was added TFA (10 mL). After 12 h, analysis byLCMS showed the reaction was complete as a mixture of the freephenylalanine carboxylic acid and the tBu ester. The crude oil waspurified by preparatory RP-HPLC with a Phenomenex Synergi 10μ Max-RP 80Å column (150×30 mm) using 10% to 90% MeCN in 0.05% aqueous TFA as theeluent. A total of 2.85 g of H-Dap-Phe-OtBu TFA salt (5.65 mmol) wasobtained as an amber oil and a total of 2.01 g of H-Dap-Phe-OH TFA salt(4.49 mmol) was obtained as a yellow oil.

To a stirred room temperature suspension of crude Dov-Cys(Trt)-Dil-OHTFA salt (0.208 g) and H-Dap-Phe-OtBu TFA salt (0.144 g, 0.369 mmol) inDMF (10 mL) was added DIEA (0.219 mL, 1.23 mmol), followed by HATU(0.234 g, 0.615 mmol). After 10 h, analysis by LCMS showed the reactionwas complete. The crude reaction mixture was diluted with saturatedsodium bicarbonate (10 mL) and extracted with EtOAc (40 mL×2). Thecombined organic fractions were washed with brine, dried over a pad ofmagnesium sulfate, filtered, and concentrated in vacuo. The crude oilwas purified by preparatory RP-HPLC with a Phenomenex Synergi 10μ Max-RP80 Å column (150×30 mm) using 10% to 90% MeCN in 0.05% aqueous TFA asthe eluent. A total of 0.509 g of enriched Dov-Cys(Trt)-Dil-Dap-Phe-OtBuwas obtained as the TFA salt.

To a room temperature solution of enriched Dov-Cys(Trt)-Dil-Dap-Phe-OtBuTFA salt (0.509) was added TFA (5 mL). The solution was heated to 60° C.and stirred for 24 h. After 24 h, analysis by LCMS showed the reactionwas complete. Volatile organics were evaporated in vacuo. The crude oilwas purified by preparatory RP-HPLC with a Phenomenex Synergi 10μ Max-RP80 Å column (150×30 mm) using 10% to 90% MeCN in 0.05% aqueous TFA asthe eluent. A total of 56.0 mg of the title compound was obtained as theTFA salt (0.065 mmol, 25%). LCMS RT=2.32 min (Method A); ESI-MS m/z750.60 [M+H]⁺; HRMS m/z 750.4460 [C₃₈H₆₃N₅O₈S+H]⁺.

Example 21(S)-2-((2R,3R)-3-((S)-1-((3R,4S,5S)-4-((S)-2-((S)-2-(dimethylamino)-3-methylbutanamido)-3-hydroxy-N-methylpropanamido)-3-methoxy-5-methylheptanoyl)pyrrolidin-2-yl)-3-methoxy-2-methylpropanamido)-3-phenylpropanoicAcid

To a stirred room temperature suspension of crude Dov-Ser(Bzl)-Dil-OHTFA salt (0.232 g) and H-Dap-Phe-OtBu TFA salt (0.196 g, 0.389 mmol) inDMF (10 mL) was added DIEA (0.326 mL, 1.83 mmol), followed by HATU(0.348, 0.914 mmol). After 6 h, analysis by LCMS showed the reaction wascomplete. The crude reaction mixture was diluted with saturated sodiumbicarbonate (10 mL) and extracted with EtOAc (40 mL×2). The combinedorganic fractions were washed with brine, dried over a pad of magnesiumsulfate, filtered, and concentrated in vacuo. The crude oil was purifiedby preparatory RP-HPLC with a Phenomenex Synergi 10μ Max-RP 80 Å column(150×30 mm) using 10% to 90% MeCN in 0.05% aqueous TFA as the eluent. Atotal of 0.378 g Dov-Ser(Bzl)-Dil-Dap-Phe-OtBu (0.380 mmol, 98%) wasobtained as the TFA salt.

To a room temperature solution of Dov-Ser(Bzl)-Dil-Dap-Phe-OtBu TFA salt(0.455 g, 0.517 mmol) in CH₂Cl₂ (5 mL) was added TFA (2 mL). After 12 h,analysis by LCMS showed the reaction was complete. Volatile organicswere evaporated in vacuo to yield crude of Dov-Ser(Bzl)-Dil-Dap-Phe-OHTFA salt that was used without further purification.

A stirred room temperature suspension of crudeDov-Ser(Bzl)-Dil-Dap-Phe-OH TFA salt from the previous step andpalladium on activated charcoal (10% Pd basis, 10.0 mg) in MeOH (10 mL)was hydrogenated. After 12 h, analysis by LCMS showed the reaction wascomplete. The crude reaction mixture was filtered over a pad ofdiatomaceous earth and the filtrate was concentrated. The crude oil waspurified by preparatory RP-HPLC with a Phenomenex Synergi 10μ Max-RP 80Å column (150×30 mm) using 10% to 90% MeCN in 0.05% aqueous TFA as theeluent. A total of 0.115 g of the title compound was obtained as the TFAsalt (0.136 mmol, 28%). LCMS RT=2.10 min (Method A); ESI-MS m/z 734.61[M+H]⁺; HRMS m/z 734.4708 [C₃₈H₆₃N₅O₉+H]⁺.

Example 22(S)-2-((2R,3R)-3-((S)-1-((3R,4S,5S)-4-((2S,3R)-2-((S)-2-(dimethylamino)-3-methylbutanamido)-3-hydroxy-N-methylbutanamido)-3-methoxy-5-methylheptanoyl)pyrrolidin-2-yl)-3-methoxy-2-methylpropanamido)-3-phenylpropanoicAcid

To a stirred room temperature suspension of crude Dov-Thr(Bzl)-Dil-OHTFA salt (0.180 g) and H-Dap-Phe-OtBu TFA salt (0.148 g, 0.294 mmol) inDMF (10 mL) was added DIEA (0.246 mL, 1.38 mmol), followed by HATU(0.262, 0.690 mmol). After 6 h, analysis by LCMS showed the reaction wascomplete. The crude reaction mixture was diluted with saturated sodiumbicarbonate (10 mL) and extracted with EtOAc (40 mL×2). The combinedorganic fractions were washed with brine, dried over a pad of magnesiumsulfate, filtered, and concentrated in vacuo. The crude oil was purifiedby preparatory RP-HPLC with a Phenomenex Synergi 10μ Max-RP 80 Å column(150×30 mm) using 10% to 90% MeCN in 0.05% aqueous TFA as the eluent. Atotal of 0.298 g Dov-Thr(Bzl)-Dil-Dap-Phe-OtBu (0.296 mmol, 78%) wasobtained as the TFA salt.

To a room temperature solution of Dov-Thr(Bzl)-Dil-Dap-Phe-OtBu TFA salt(0.298 g, 0.296 mmol) in CH₂Cl₂ (5 mL) was added TFA (2 mL). After 10 h,analysis by LCMS showed the reaction was complete. Volatile organicswere evaporated in vacuo to yield crude of Dov-Thr(Bzl)-Dil-Dap-Phe-OHTFA salt that was used without further purification.

A stirred room temperature suspension of crudeDov-Thr(Bzl)-Dil-Dap-Phe-OH TFA salt from the previous step andpalladium on activated charcoal (10% Pd basis, 10.0 mg) in MeOH (10 mL)was hydrogenated. After 12 h, analysis by LCMS showed the reaction wascomplete. The crude reaction mixture was filtered over a pad ofdiatomaceous earth and the filtrate was concentrated. The crude oil waspurified by preparatory RP-HPLC with a Phenomenex Synergi 10μ Max-RP 80Å column (150×30 mm) using 10% to 90% MeCN in 0.05% aqueous TFA as theeluent. A total of 0.120 g of the title compound was obtained as the TFAsalt (0.139 mmol, 47%). LCMS RT=2.22 min (Method A); ESI-MS m/z 748.62[M+H]⁺; HRMS m/z 748.4842 [C₃₉H₆₅N₅O₉+H]⁺.

Example 23 (S)-methyl2-((2R,3R)-3-((S)-1-((3R,4S,5S)-4-((S)-2-((S)-2-(dimethylamino)-3-hydroxypropanamido)-N,3-dimethylbutanamido)-3-methoxy-5-methylheptanoyl)pyrrolidin-2-yl)-3-methoxy-2-methylpropanamido)-3-phenylpropanoate

A stirred room temperature suspension ofN,N-dimethylSer(Bzl)-Val-Dil-Dap-Phe-OMe TFA salt (50.0 mg, 0.053 mmol)and palladium on activated charcoal (10% Pd basis, 10 mg) in MeOH (10mL) was hydrogenated. After 10 h, analysis by LCMS showed the reactionwas complete. The crude reaction mixture was filtered over a pad ofdiatomaceous earth and the filtrate was concentrated. The crude oil waspurified by preparatory RP-HPLC with a Phenomenex Synergi 10μ Max-RP 80Å column (150×30 mm) using 10% to 90% MeCN in 0.05% aqueous TFA as theeluent. A total of 8.00 mg of the title compound was obtained as the TFAsalt (0.009 mmol, 18%). LCMS RT=2.38 min (Method A); ESI-MS m/z 748.57[M+H]⁺; HRMS m/z 748.4849 [C₃₉H₆₅N₅O₉+H]⁺.

Example 24 (S)-methyl2-((2R,3R)-3-((S)-1-((3R,4S,5S)-4-((S)-2-((2S,3R)-2-(dimethylamino)-3-hydroxybutanamido)-N,3-dimethylbutanamido)-3-methoxy-5-methylheptanoyl)pyrrolidin-2-yl)-3-methoxy-2-methylpropanamido)-3-phenylpropanoate

The title compound may be prepared using methods analogous to thosedescribed in the Examples and general synthetic schemes.

Example 25 (S)-methyl2-((2R,3R)-3-((S)-1-((3R,4S,5S)-4-((2S,3S)-3-amino-2-((S)-2-(dimethylamino)-3-methylbutanamido)-N-methylbutanamido)-3-methoxy-5-methylheptanoyl)pyrrolidin-2-yl)-3-methoxy-2-methylpropanamido)-3-phenylpropanoate

To a stirred room temperature solution of Dov-Abu(3-N₃)-Dil-Dap-Phe-OMeTFA salt (10 mg, 0.011 mmol) in THF (0.10 mL) was addedtrimethylphosphine in THF (1 M, 0.022 mL, 0.022 mmol). After 4 h,analysis by LCMS showed the reaction was complete and H₂O (0.05 mL) wasadded to the reaction mixture. The crude reaction mixture was purifiedby preparatory RP-HPLC with a Phenomenex Synergi 10μ Max-RP 80 Å column(150×30 mm) using 10% to 90% MeCN in 0.05% aqueous TFA as the eluent. Atotal of 6.00 mg of the title compound was obtained as the TFA salt(0.007 mmol, 62%). LCMS RT=2.12 min (Method A); ESI-MS m/z 761.63[M+H]⁺; HRMS m/z 761.5159 [C₄₀H₆₈N₆O₈+H]⁺.

Example 26(S)-2-((2R,3R)-3-((S)-1-((3R,4S,5S)-4-((2S,3S)-3-amino-2-((S)-2-(dimethylamino)-3-methylbutanamido)-N-methylbutanamido)-3-methoxy-5-methylheptanoyl)pyrrolidin-2-yl)-3-methoxy-2-methylpropanamido)-3-phenylpropanoicAcid

To a stirred room temperature solution of Dov-Abu(3-N₃)-Dil-Dap-Phe-OHTFA salt (10 mg, 0.011 mmol) in THF (0.10 mL) was addedtrimethylphosphine in THF (1 M, 0.022 mL, 0.022 mmol). After 4 h,analysis by LCMS showed the reaction was complete and H₂O (0.05 mL) wasadded to the reaction mixture. The crude reaction mixture was purifiedby preparatory RP-HPLC with a Phenomenex Synergi 10μ Max-RP 80 Å column(150×30 mm) using 10% to 90% MeCN in 0.05% aqueous TFA as the eluent. Atotal of 2.00 mg of the title compound was obtained as the TFA salt(0.002 mmol, 21%). LCMS RT=2.02 min (Method A); ESI-MS m/z 747.65[M+H]⁺; HRMS m/z 747.5008 [C₃₉H₆₆N₆O₈+H]⁺.

Example 27(2S,3S)-3-azido-2-((S)-2-(dimethylamino)-3-methylbutanamido)-N-((3R,4S,5S)-3-methoxy-1-((S)-2-((1R,2R)-1-methoxy-2-methyl-3-oxo-3-(phenethylamino)propyl)pyrrolidin-1-yl)-5-methyl-1-oxoheptan-4-yl)-N-methylbutanamide

To a stirred 25° C. solution of Boc-Dap-OH dicyclohexylamine salt (6.47g, 13.8 mmol) and phenethylamine (3.914 g, 15.19 mmol) in CH₂Cl₂ (20 mL)was added DIEA (8.76 mL, 55.2 mmol), followed by DEPC (3.12 mL, 20.7mmol). After 8 h, analysis by LCMS showed the reaction was complete. Thevolatile organic were evaporated in vacuo to give crude product that wasused without further purification. A total of 4.04 g ofBoc-Dap-phenethylamine (10.3 mmol) was obtained. LCMS RT=3.00 min(Method A); ESI-MS m/z 391.37 [M+H]⁺.

To a stirred 25° C. solution of Boc-Dap-phenethylamine (4.04 g, 10.3mmol) in CH₂Cl₂ (15.0 mL) was added TFA (15.0 mL). After 14 h, analysisby LCMS showed the reaction was complete. The volatile organic wereevaporated in vacuo an the crude oil was purified by preparatory RP-HPLCwith a Phenomenex Synergi 10μ Max-RP 80 Å column (150×30 mm) using 10%to 90% MeCN in 0.05% aqueous TFA as the eluent. A total of 2.51 g ofH-Dap-phenethylamine (6.21 mmol) was obtained as the TFA salt. LCMSRT=1.72 min (Method A); ESI-MS m/z 291.29 [M+H]⁺.

To a stirred room temperature solution of Dov-Abu(3-N₃)-Dil-OH TFA salt(0.300 g, 0.526 mmol) and H-Dap-phenethylamine TFA salt (0.191 g, 0.471mmol) in DMF (5 mL) was added DIEA (0.343 mL, 1.97 mmol), followed bythe addition of HATU (0.501 g, 1.31 mmol). After 10 h, analysis by LCMSshowed the reaction was complete. The crude reaction was diluted withsaturated sodium bicarbonate (10 mL) and extracted with EtOAc (20 mL×2).The combined organic fractions were washed with brine, dried usingmagnesium sulfate, filtered, and concentrated in vacuo. The crude oilwas purified by preparatory RP-HPLC with a Phenomenex Synergi 10μ Max-RP80 Å column (150×30 mm) using 10% to 90% MeCN in 0.05% aqueous TFA asthe eluent. A total of 196 mg of the title compound was obtained as theTFA salt (0.233 mmol, 49%). LCMS RT=2.45 min (Method A); ESI-MS m/z729.55 [M+H]⁺; HRMS m/z 729.5030 [C₃₈H₆₄N₈O₆+H]⁺.

Example 28(2S,3S)-3-azido-2-((S)-2-(dimethylamino)-3-methylbutanamido)-N-((3R,4S,5S)-1-((S)-2-((1R,2R)-3-(((1S,2R)-1-hydroxy-1-phenylpropan-2-yl)amino)-1-methoxy-2-methyl-3-oxopropyl)pyrrolidin-1-yl)-3-methoxy-5-methyl-1-oxoheptan-4-yl)-N-methylbutanamide

To a stirred room temperature solution of Boc-Dap-OH dicyclohexylaminesalt (10.0 g, 0.021 mol) and (1R,2S)-(−)-Norephedrine (3.87 g, 0.026mol) in CH₂Cl₂ (20 mL) was added DIEA (11.4 mL, 0.064 mol) and DEPC(6.44 mL, 0.043 mol). After 14 h, analysis by LCMS showed the reactionwas complete. To the crude reaction mixture was added 0.1 M HCl (20.0mL). The organic layer was separated, dried over a pad of magnesiumsulfate, filtered, and concentrated in vacuo to yield crude product. Atotal of 7.92 g of Boc-Dap-(1R,2S)-(−)-Norephedrine (18.8 mmol, 88%) wasobtained. LCMS RT=2.25 min (Method A); ESI-MS m/z 421.31 [M+H]⁺.

To a stirred room temperature solution ofBoc-Dap-(1R,2S)-(−)-Norephedrine (7.92 g, 18.8 mmol) in CH₂Cl₂ (10 mL)was added TFA (10 mL). After 10 h, analysis by LCMS showed the reactionwas complete. The crude oil was purified by preparatory RP-HPLC with aPhenomenex Synergi 10μ Max-RP 80 Å column (150×30 mm) using 10% to 90%MeCN in 0.05% aqueous TFA as the eluent. A total of 5.00 g ofH-Dap-(1R,2S)-(−)-Norephedrine (11.5 mmol, 61%) was prepared as the TFAsalt. LCMS RT=1.10 min (Method A); ESI-MS m/z 321.33 [M+H]⁺.

To a stirred room temperature solution of Dov-Abu(3-N₃)-Dil-OH TFA salt(0.300 g, 0.526 mmol) and H-Dap-(1R,2S)-(−)-Norephedrine TFA salt (0.211g, 0.486 mmol) in DMF (5 mL) was added DIEA (0.351 mL, 1.97 mmol),followed by the addition of HATU (0.501 g, 1.31 mmol). After 10 h,analysis by LCMS showed the reaction was complete. The crude reactionwas diluted with saturated sodium bicarbonate (10 mL) and extracted withEtOAc (20 mL×2). The combined organic fractions were washed with brine,dried using magnesium sulfate, filtered, and concentrated in vacuo. Thecrude oil was purified by preparatory RP-HPLC with a Phenomenex Synergi10μ Max-RP 80 Å column (150×30 mm) using 10% to 90% MeCN in 0.05%aqueous TFA as the eluent. A total of 128 mg of the title compound wasobtained as the TFA salt (0.147 mmol, 30%). LCMS RT=1.95 min (Method A);ESI-MS m/z 759.65 [M+H]⁺; HRMS m/z 759.5121 [C₃₉H₆₆N₈O₇+H]⁺.

Example 29(2S,3S)-3-azido-N-((3R,4S,5S)-1-((S)-2-((1R,2R)-3-((4-chlorophenethyl)amino)-1-methoxy-2-methyl-3-oxopropyl)pyrrolidin-1-yl)-3-methoxy-5-methyl-1-oxoheptan-4-yl)-2-((S)-2-(dimethylamino)-3-methylbutanamido)-N-methylbutanamide

To a stirred room temperature solution of Boc-Dap-OH dicyclohexylaminesalt (5.00 g, 10.7 mmol) and 2-(4-chlorophenyl)ethylamine (1.85 g, 11.7mmol) in CH₂Cl₂ (20 mL) was added DIEA (6.78 mL, 42.7 mmol) and DEPC(2.41 mL, 16.0 mmol). After 10 h, analysis by LCMS showed the reactionwas complete. The volatile organic were evaporated in vacuo to givecrude product that was used without further purification. A total of4.25 g of Boc-Dap-2-(4-chlorophenyl)ethylamine (10.0 mmol, 94%) wasobtained. LCMS RT=3.10 min (Method A); ESI-MS m/z 425.32 [M+H]⁺.

To a stirred room temperature solution ofBoc-Dap-2-(4-chlorophenyl)ethylamine (4.25 g, 10.0 mmol) in CH₂Cl₂ (15mL) was added TFA (15 mL). After 10 h, analysis by LCMS showed thereaction was complete. The crude oil was purified by preparatory RP-HPLCwith a Phenomenex Synergi 10μ Max-RP 80 Å column (150×30 mm) using 10%to 90% MeCN in 0.05% aqueous TFA as the eluent. A total of 2.27 g ofH-Dap-2-(4-chlorophenyl)ethylamine (5.18 mmol, 52%) was prepared as theTFA salt. LCMS RT=2.05 min (Method A); ESI-MS m/z 325.24 [M+H]⁺.

To a stirred room temperature solution of Dov-Abu(3-N₃)-Dil-OH TFA salt(0.526 g, 0.923 mmol) and H-Dap-2-(4-chlorophenyl)ethylamine TFA salt(0.347 g, 0.792 mmol) in DMF (10 mL) was added DIEA (0.602 mL, 3.46mmol), followed by the addition of HATU (0.878 g, 2.30 mmol). After 10h, analysis by LCMS showed the reaction was complete. The crude reactionwas diluted with saturated sodium bicarbonate (10 mL) and extracted withEtOAc (20 mL×2). The combined organic fractions were washed with brine,dried using magnesium sulfate, filtered, and concentrated in vacuo. Thecrude oil was purified by preparatory RP-HPLC with a Phenomenex Synergi10μ Max-RP 80 Å column (150×30 mm) using 10% to 90% MeCN in 0.05%aqueous TFA as the eluent. A total of 596 mg of the title compound wasobtained as the TFA salt (0.680 mmol, 85%). LCMS RT=2.38 min (Method A);ESI-MS m/z 763.71 [M+H]⁺; HRMS m/z 763.4633 [C₃₈H₆₃N₈O₆Cl+H]⁺.

Example 30(2S,3S)-3-azido-N-((3R,4S,5S)-1-((S)-2-((1R,2R)-3-((2-chlorophenethyl)amino)-1-methoxy-2-methyl-3-oxopropyl)pyrrolidin-1-yl)-3-methoxy-5-methyl-1-oxoheptan-4-yl)-2-((S)-2-(dimethylamino)-3-methylbutanamido)-N-methylbutanamide

To a stirred room temperature solution of Boc-Dap-OH dicyclohexylaminesalt (5.00 g, 10.7 mmol) and 2-(2-chlorophenyl)ethylamine (1.85 g, 11.7mmol) in CH₂Cl₂ (10 mL) was added DIEA (4.76 mL, 26.7 mmol) and DEPC(2.41 mL, 16.0 mmol). After 10 h, analysis by LCMS showed the reactionwas complete. The volatile organic were evaporated in vacuo to givecrude product that was used without further purification. A total of3.98 g of Boc-Dap-2-(2-chlorophenyl)ethylamine (9.37 mmol, 88%) wasobtained. LCMS RT=3.04 min (Method A); ESI-MS m/z 425.23 [M+H]⁺.

To a stirred room temperature solution ofBoc-Dap-2-(2-chlorophenyl)ethylamine (3.98 g, 9.37 mmol) in CH₂Cl₂ (5mL) was added TFA (5 mL). After 10 h, analysis by LCMS showed thereaction was complete. The crude oil was purified by preparatory RP-HPLCwith a Phenomenex Synergi 10μ Max-RP 80 Å column (150×30 mm) using 10%to 90% MeCN in 0.05% aqueous TFA as the eluent. A total of 2.87 g ofH-Dap-2-(2-chlorophenyl)ethylamine (6.55 mmol, 70%) was prepared as theTFA salt. LCMS RT=1.83 min (Method A); ESI-MS m/z 325.22 [M+H]⁺.

To a stirred room temperature solution of Dov-Abu(3-N₃)-Dil-OH TFA salt(0.450 g, 0.789 mmol) and H-Dap-2-(2-chlorophenyl)ethylamine TFA salt(0.320 g, 0.731 mmol) in DMF (10 mL) was added DIEA (0.515 mL, 2.96mmol), followed by the addition of HATU (0.751 g, 1.97 mmol). After 10h, analysis by LCMS showed the reaction was complete. The crude reactionmixture was purified by preparatory RP-HPLC with a Phenomenex Synergi10μ Max-RP 80 Å column (150×30 mm) using 10% to 90% MeCN in 0.05%aqueous TFA as the eluent. A total of 253 mg of the title compound wasobtained as the TFA salt (0.289 mmol, 32% based on RSM). LCMS RT=1.26min (Method B); ESI-MS m/z 763.60 [M+H]⁺; HRMS m/z 763.4632[C₃₈H₆₃N₈O₆Cl+H]⁺.

Example 31(2S,3S)-3-amino-2-((S)-2-(dimethylamino)-3-methylbutanamido)-N-((3R,4S,5S)-3-methoxy-1-((S)-2-((1R,2R)-1-methoxy-2-methyl-3-oxo-3-(phenethylamino)propyl)pyrrolidin-1-yl)-5-methyl-1-oxoheptan-4-yl)-N-methylbutanamide

To a stirred room temperature solution of Dov-Abu(3-N₃)-Dil-Dap-PE TFAsalt (25 mg, 0.030 mmol) in THF (0.5 mL) was added trimethylphosphine inTHF (1 M, 0.045 mL, 0.045 mmol). After 1 h, analysis by LCMS showed thereaction was complete. The crude reaction mixture was purified bypreparatory RP-HPLC with a Phenomenex Synergi 10μ Max-RP 80 Å column(150×30 mm) using 5% to 95% MeCN in 0.1% aqueous formic acid as theeluent. A total of 9.0 mg of the title compound was obtained as theformic acid salt (0.012 mmol, 41%). LCMS RT=1.02 min (Method B); ESI-MSm/z 703.71 [M+H]⁺; HRMS m/z 703.5117 [C₃₈H₆₆N₆O₆+H]⁺.

Example 32(2S,3S)-3-amino-2-((S)-2-(dimethylamino)-3-methylbutanamido)-N-((3R,4S,5S)-1-((S)-2-((1R,2R)-3-(((1S,2R)-1-hydroxy-1-phenylpropan-2-yl)amino)-1-methoxy-2-methyl-3-oxopropyl)pyrrolidin-1-yl)-3-methoxy-5-methyl-1-oxoheptan-4-yl)-N-methylbutanamide

To a stirred room temperature solution ofDov-Abu(3-N₃)-Dil-Dap-Norephedrine TFA salt (27 mg, 0.031 mmol) in THF(0.3 mL) was added trimethylphosphine in THF (1 M, 0.046 mL, 0.046mmol). After 2 h, analysis by LCMS showed the reaction was complete. Thecrude reaction mixture was purified by preparatory RP-HPLC with aPhenomenex Synergi 10μ Max-RP 80 Å column (150×30 mm) using 10% to 90%MeCN in 0.05% aqueous trifluoroacetic acid as the eluent. A total of 6.8mg of the title compound was obtained as the TFA salt (0.008 mmol, 26%).LCMS RT=1.95 min (Method B); ESI-MS m/z 733.72 [M+H]⁺; HRMS m/z 733.5227[C₃₉H₆₈N₆O₇+H]⁺.

Example 33(2S,3S)-3-amino-N-((3R,4S,5S)-1-((S)-2-((1R,2R)-3-((4-chlorophenethyl)amino)-1-methoxy-2-methyl-3-oxopropyl)pyrrolidin-1-yl)-3-methoxy-5-methyl-1-oxoheptan-4-yl)-2-((S)-2-(dimethylamino)-3-methylbutanamido)-N-methylbutanamide

To a stirred room temperature solution ofDov-Abu(3-N₃)-Dil-Dap-2-(4-chlorophenyl)ethylamine TFA salt (117 mg,0.133 mmol) in THF (0.3 mL) was added trimethylphosphine in THF (1 M,0.2 mL, 0.2 mmol). After 2 h, analysis by LCMS showed the reaction wascomplete. The crude reaction mixture was purified by preparatory RP-HPLCwith a Phenomenex Synergi 10μ Max-RP 80 Å column (150×30 mm) using 5% to95% MeCN in 0.1% aqueous formic acid as the eluent. A total of 9.0 mg ofthe title compound was obtained as the formic acid salt (0.011 mmol,9%). LCMS RT=0.97 min (Method B); ESI-MS m/z 737.51 [M+H]⁺; HRMS m/z737.4731 [C₃₈H₆₅N₆O₆Cl+H]⁺.

Example 34(2S,3S)-3-amino-N-((3R,4S,5S)-1-((S)-2-((1R,2R)-3-((2-chlorophenethyl)amino)-1-methoxy-2-methyl-3-oxopropyl)pyrrolidin-1-yl)-3-methoxy-5-methyl-1-oxoheptan-4-yl)-2-((S)-2-(dimethylamino)-3-methylbutanamido)-N-methylbutanamide

To a stirred room temperature solution ofDov-Abu(3-N₃)-Dil-Dap-2-(2-chlorophenyl)ethylamine formic acid salt (46mg, 0.057 mmol) in THF (1.0 mL) was added trimethylphosphine in THF (1M, 0.085 mL, 0.085 mmol). After 1 h, the crude reaction mixture waspurified by preparatory RP-HPLC with a Phenomenex Synergi 10μ Max-RP 80Å column (150×30 mm) using 5% to 95% MeCN in 0.1% aqueous formic acid asthe eluent. A total of 13.6 mg of the title compound was obtained as theformic acid salt (0.017 mmol, 30%). LCMS RT=1.03 min (Method B); ESI-MSm/z 737.57 [M+H]⁺; HRMS m/z 737.4731 [C₃₈H₆₅N₆O₆Cl+H]⁺.

Example 35(S)-4-amino-2-((S)-2-(dimethylamino)-3-methylbutanamido)-N-((3R,4S,5S)-3-methoxy-1-((S)-2-((1R,2R)-1-methoxy-2-methyl-3-oxo-3-(phenethylamino)propyl)pyrrolidin-1-yl)-5-methyl-1-oxoheptan-4-yl)-N-methylbutanamide

To a stirred room temperature solution of Dov-Dab(Fmoc)-Dil-OH TFA salt(0.450 g, 0.587 mmol) and H-Dap-phenethylamine TFA salt (0.200 g, 0.495mmol) in DMF (15 mL) was added DIEA (0.360 mL, 2.07 mmol), followed bythe addition of HATU (0.526 g, 1.38 mmol). After 10 h, analysis by LCMSshowed the reaction was complete. The crude reaction mixture was dilutedwith saturated sodium bicarbonate (10 mL) and extracted with EtOAc (20mL×2). The combined organic fractions were washed with brine, driedusing magnesium sulfate, filtered, and concentrated in vacuo. The crudeoil was purified by preparatory RP-HPLC with a Phenomenex Synergi 10μMax-RP 80 Å column (150×30 mm) using 10% to 90% MeCN in 0.1% aqueousformic acid as the eluent. A total of 276 mg ofDov-Dab(Fmoc)-Dil-Dap-phenethylamine (0.266 mmol, 54%) was obtained asthe formic acid salt. LCMS RT=1.37 min (Method B); ESI-MS m/z 925.48[M+H]⁺.

To a stirred room temperature solution ofDov-Dab(Fmoc)-Dil-Dap-phenethylamine (0.276 g, 0.266 mmol) inacetonitrile (10 mL) was added piperidine (5 mL). After 10 h, analysisby LCMS showed the reaction was complete. To the crude reaction mixturewas added hexanes.

The acetonitrile layer was concentrated in vacuo. The crude oil waspurified by preparatory RP-HPLC with a Phenomenex Synergi 10μ Max-RP 80Å column (150×30 mm) using 10% to 90% MeCN in 0.1% aqueous formic acidas the eluent. A total of 98.0 mg of the title compound was obtained asa formic acid salt (0.120 mmol, 45%). LCMS RT=1.02 min (Method B);ESI-MS m/z 703.78 [M+H]⁺; HRMS m/z 703.5117 [C₃₈H₆₆N₆O₆+H]⁺.

Example 36(S)-4-amino-2-((S)-2-(dimethylamino)-3-methylbutanamido)-N-((3R,4S,5S)-1-((S)-2-((1R,2R)-3-(((1S,2R)-1-hydroxy-1-phenylpropan-2-yl)amino)-1-methoxy-2-methyl-3-oxopropyl)pyrrolidin-1-yl)-3-methoxy-5-methyl-1-oxoheptan-4-yl)-N-methylbutanamide

To a stirred room temperature solution of Dov-Dab(Fmoc)-Dil-OH TFA salt(0.459 g, 0.599 mmol) and H-Dap-(1R,2S)-(−)-Norephedrine TFA salt (0.225g, 0.518 mmol) in DMF (15 mL) was added DIEA (0.376 mL, 2.11 mmol),followed by the addition of HATU (0.536 g, 1.41 mmol). After 10 h,analysis by LCMS showed the reaction was complete. The crude reactionmixture was diluted with saturated sodium bicarbonate (10 mL) andextracted with EtOAc (20 mL×2). The combined organic fractions werewashed with brine, dried using magnesium sulfate, filtered, andconcentrated in vacuo. The crude oil was purified by preparatory RP-HPLCwith a Phenomenex Synergi 10μ Max-RP 80 Å column (150×30 mm) using 10%to 90% MeCN in 0.1% aqueous formic acid as the eluent. A total of 449 mgof Dov-Dab(Fmoc)-Dil-Dap-(1R,2S)-(−)-Norephedrine (0.420 mmol, 81%) wasobtained as the formic acid salt. LCMS RT=1.35 min (Method B); ESI-MSm/z 955.74 [M+H]⁺.

To a stirred room temperature solution ofDov-Dab(Fmoc)-Dil-Dap-(1R,2S)-(−)-Norephedrine formic acid salt (0.449g, 0.420 mmol) in acetonitrile (10 mL) was added piperidine (5 mL).After 10 h, analysis by LCMS showed the reaction was complete. To thecrude reaction mixture was added hexanes. The acetonitrile layer wasconcentrated in vacuo. The crude oil was purified by preparatory RP-HPLCwith a Phenomenex Synergi 10μ Max-RP 80 Å column (150×30 mm) using 10%to 90% MeCN in 0.1% aqueous formic acid as the eluent. A total of 53.0mg of the title compound was obtained as a formic acid salt (0.068 mmol,14%). LCMS RT=0.79 min (Method B); ESI-MS m/z 733.71 [M+H]⁺; HRMS m/z733.5227 [C₃₉H₆₈N₆O₇+H]⁺.

Example 37(S)-4-amino-N-((3R,4S,5S)-1-((S)-2-((1R,2R)-3-((4-chlorophenethyl)amino)-1-methoxy-2-methyl-3-oxopropyl)pyrrolidin-1-yl)-3-methoxy-5-methyl-1-oxoheptan-4-yl)-2-((S)-2-(dimethylamino)-3-methylbutanamido)-N-methylbutanamide

To a stirred room temperature solution of Dov-Dab(Fmoc)-Dil-OH TFA salt(0.255 g, 0.333 mmol) and H-Dap-2-(4-chlorophenyl)ethylamine TFA salt(0.127 g, 0.290 mmol) in DMF (10 mL) was added DIEA (0.204 mL, 1.17mmol), followed by the addition of HATU (0.298 g, 0.781 mmol). After 10h, analysis by LCMS showed the reaction was complete. The crude reactionmixture was diluted with saturated sodium bicarbonate (10 mL) andextracted with EtOAc (20 mL×2). The combined organic fractions werewashed with brine, dried using magnesium sulfate, filtered, andconcentrated in vacuo. The crude oil was purified by preparatory RP-HPLCwith a Phenomenex Synergi 10μ Max-RP 80 Å column (150×30 mm) using 10%to 90% MeCN in 0.1% aqueous formic acid as the eluent. A total of 190 mgof Dov-Dab(Fmoc)-Dil-Dap -2-(4-chlorophenyl)ethylamine (0.177 mmol, 61%)was obtained as the formic acid salt. LCMS RT=1.49 min (Method B);ESI-MS m/z 959.62 [M+H]⁺.

To a stirred room temperature solution ofDov-Dab(Fmoc)-Dil-Dap-2-(4-chlorophenyl)ethylamine formic acid salt(0.190 g, 0.177 mmol) in acetonitrile (5 mL) was added piperidine (5mL). After 10 h, analysis by LCMS showed the reaction was complete. Tothe crude reaction mixture was added hexanes. The acetonitrile layer wasconcentrated in vacuo. The crude oil was purified by preparatory RP-HPLCwith a Phenomenex Synergi 10μ Max-RP 80 Å column (150×30 mm) using 10%to 90% MeCN in 0.1% aqueous formic acid as the eluent. A total of 118 mgof the title compound was obtained as a formic acid salt (0.151 mmol,85%). LCMS RT=1.06 min (Method B); ESI-MS m/z 737.55 [M+H]⁺; HRMS m/z737.4729 [C₃₈H₆₅N₆O₆Cl+H]⁺.

Example 38(S)-4-amino-N-((3R,4S,5S)-1-((S)-2-((1R,2R)-3-((2-chlorophenethyl)amino)-1-methoxy-2-methyl-3-oxopropyl)pyrrolidin-1-yl)-3-methoxy-5-methyl-1-oxoheptan-4-yl)-2-((S)-2-(dimethylamino)-3-methylbutanamido)-N-methylbutanamide

To a stirred room temperature solution of Dov-Dab(Fmoc)-Dil-OH TFA salt(0.306 g, 0.399 mmol) and H-Dap-2-(2-chlorophenyl)ethylamine TFA salt(0.170 g, 0.388 mmol) in DMF (5 mL) was added DIEA (0.300 g, 400 μL,2.29 mmol), followed by the addition of HATU (360 mg, 0.944 mmol). After10 h, analysis by LCMS showed the reaction was complete. The crudereaction was diluted with saturated sodium bicarbonate (10 mL) andextracted with EtOAc (10 mL×3). The combined organic fractions werewashed with brine, dried over a pad of magnesium sulfate, filtered, andconcentrated in vacuo. The crude oil was purified by preparatory RP-HPLCwith a Phenomenex Gemini 10 Max-RP 110 Å column (150×30 mm) using 10% to90% MeCN in 0.1% aqueous NH₄OH as the eluent. A total of 115 mg ofDov-Dab(Fmoc)-Dil-Dap -2-(2-chlorophenyl)ethylamine (0.120 mmol, 26%)was obtained as a white solid. LCMS RT=1.40 min (Method B); ESI-MS m/z959.75 [M+H]⁺.

To a stirred room temperature solution ofDov-Dab(Fmoc)-Dil-Dap-2-(2-chlorophenyl)ethylamine (0.115 g, 0.120 mmol)in acetonitrile (10 mL) was added piperidine (2 mL). After 3 h, analysisby LCMS showed the reaction was complete. To the crude reaction mixturewas added hexanes. The acetonitrile layer was concentrated in vacuo. Thecrude oil was purified by preparatory RP-HPLC with a Phenomenex Synergi10μ Max-RP 80 Å column (150×30 mm) using 10% to 90% MeCN in 0.1% aqueousNH₄OH as the eluent. A total of 30.0 mg of the title compound wasobtained as a white solid (0.041 mmol, 34%). LCMS RT=1.15 min (MethodB); ESI-MS m/z 737.68 [M+H]⁺; HRMS m/z 737.4729 [C₃₈H₆₅N₆O₆Cl+H]⁺.

Example 39 methyl((2R,3R)-3-((S)-1-((3R,4S,5S)-4-((S)-2-((S)-2-(dimethylamino)-3-methylbutanamido)-N-methylpent-4-ynamido)-3-methoxy-5-methylheptanoyl)pyrrolidin-2-yl)-3-methoxy-2-methylpropanoyl)-L-phenylalaninate

To a stirred 25° C. solution of Boc-propargylGly-OH (1.00 g, 4.69 mmol)and H-Dil-OtBu HCl salt (1.15 g, 3.90 mmol) in EtOAc (10 mL) was addedDIEA (1.49 mL, 9.38 mmol). The solution was cooled to (0° C.) andstirred for 20 min and an additional portion of DIEA (1.49 mL, 9.38mmol) was added and the reaction mixture stirred at 0° C. 20 min. ThenCMPI (1.80 g, 7.04 mmol) was added to the reaction mixture which wasallowed to warm to room temperature and stirred for 12 h. The crudereaction mixture was washed with 0.1 M HCl (20 mL×2), followed by brine(20 mL×2). The organic fraction was dried over anhydrous magnesiumsulfate, filtered, and concentrated in vacuo to give the crude product.A total of 2.05 g of Boc-propargylGly-Dil-OtBu was obtained (4.50 mmol,96%). LCMS RT=3.46 min (Method A); ESI-MS m/z 455.42 [M+H]⁺.

To a stirred 25° C. solution of Boc-propargylGly-Dil-OtBu (2.05 g, 4.50mmol) in CH₂Cl₂ (6 mL) was added TFA (6 mL). After 14 h, analysis byLCMS showed the reaction was complete. The volatile organics wereconcentrated in vacuo to give crude product that was used withoutfurther purification. A total of 1.30 g of H-propargylGly-Dil-OH wasobtained as the TFA salt (3.16 mmol, 70%).

To a stirred 25° C. solution of Dov (1.27 g, 8.71 mmol) in DMF (10 mL)was added DIEA (2.33 mL, 13.1 mmol), followed by HATU (3.32 g, 8.71mmol). After 10 min, a solution of H-propargylGly-Dil-OH TFA salt (1.30g, 3.16 mmol) in DMF was added to the reaction. After 8 h, analysis byLCMS showed the reaction was complete. The crude oil was purified bypreparatory RP-HPLC with a Phenomenex Synergi 10μ Max-RP 80 Å column(150×30 mm) using 10% to 90% MeCN in 0.05% aqueous TFA as the eluent. Atotal of 455 mg of Dov-propargyGly-Dil-OH was obtained as the TFA salt(0.844 mmol, 27% yield). LCMS RT=1.65 min (Method A); ESI-MS m/z 425.95[M+H]⁺.

To a stirred room temperature solution of Dov-propargylGly-Dil-OH TFAsalt (0.198 g, 0.367 mmol) and H-Dap-Phe-OMe TFA salt (0.156 g, 0.338)in DMF (10 mL) was added DIEA (0.222 mL, 1.40 mmol), followed by theaddition of HATU (0.355 g, 0.931 mmol). After 10 h, analysis by LCMSshowed the reaction was complete. The crude reaction was diluted withsaturated sodium bicarbonate (10 mL) and extracted with EtOAc (20 mL×2).The combined organic fractions were washed with brine, dried usingmagnesium sulfate, filtered, and concentrated in vacuo. The crude oilwas purified by preparatory RP-HPLC with a Phenomenex Synergi 10μ Max-RP80 Å column (150×30 mm) using 10% to 90% MeCN in 0.05% aqueous TFA asthe eluent. A total of 10.0 mg of the title compound was obtained as theTFA salt (0.012 mmol, 3%). LCMS RT=2.50 min (Method A); ESI-MS m/z756.44 [M+H]⁺; HRMS m/z 756.4902 [C₄₁H₆₅N₅O₈+H]⁺.

Example 40(2S,3S)—N-((3R,4S,5S)-1-((S)-2-((1R,2R)-3-(((S)-1-amino-1-oxo-3-phenylpropan-2-yl)amino)-1-methoxy-2-methyl-3-oxopropyl)pyrrolidin-1-yl)-3-methoxy-5-methyl-1-oxoheptan-4-yl)-3-azido-2-((S)-2-(dimethylamino)-3-methylbutanamido)-N-methylbutanamide

To a stirred room temperature solution of Dov-Abu(3-N₃)-Dil-Dap-Phe-OHformic acid salt (42.1 mg, 0.051 mmol), ammonium chloride (7.9 mg, 0.148mmol), TBTU (52.5 mg, 0.163 mmol) in DMF (0.2 mL) was added Hunig's base(0.045 mL, 0.258 mmol). After 16 h, analysis by LCMS showed the reactionwas complete. The crude reaction mixture was purified by preparatoryRP-HPLC with a Phenomenex Gemini-NX 10μ C-18 110 Å column (150×30 mm)using 5% to 95% MeCN in 0.1% aqueous ammonium hydroxide as the eluent. Atotal of 13.4 mg of the title compound was obtained (0.017 mmol, 33%).LCMS RT=1.05 min (Method B); ESI-MS m/z 772.61 [M+H]⁺; HRMS m/z 772.5078[C₃₉H₆₅N₉O₇+H]⁺.

Example 41(2S,3S)-3-azido-2-((S)-2-(dimethylamino)-3-methylbutanamido)-N-((3R,4S,5S)-3-methoxy-1-((S)-2-((1R,2R)-1-methoxy-2-methyl-3-oxo-3-((2-(pyridin-2-yl)ethyl)amino)propyl)pyrrolidin-1-yl)-5-methyl-1-oxoheptan-4-yl)-N-methylbutanamide

To a stirred 23° C. solution of Dov-Abu(3-N₃)-Dil-OH TFA salt (0.300 g,0.526 mmol) and H-Dap-2-(2-pyridyl)ethylamine TFA salt (0.287 g, 0.709mmol) in DMF (10 mL) was added DIEA (343 μL, 1.97 mmol) followed by theaddition of HATU (0.501 g, 1.31 mmol). After 10 h, analysis by LCMSshowed the reaction was complete. The crude reaction was diluted withsaturated sodium bicarbonate (10 mL) and extracted with EtOAc (20 mL×2).The combined organic fractions were washed with brine, dried over a padof magnesium sulfate, filtered, and concentrated in vacuo. The crude oilwas purified by preparatory RP-HPLC with a Phenomenex Gemini-NX 10μ C-18110 Å column (150×30 mm) using 10% to 90% MeCN in 0.1% aqueous NH₄OH asthe eluent. A total of 139 mg of the title compound was obtained (0.190mmol, 29%). LCMS RT=0.916 min (Method B); ESI-MS m/z 730.64 [M+H]⁺; HRMSm/z 730.4985 [C₃₇H₆₃N₉O₆+H]⁺.

Example 42(2S,3S)-3-azido-N-((3R,4S,5S)-1-((S)-2-((1R,2R)-3-(((S)-1-(tert-butylamino)-1-oxo-3-phenylpropan-2-yl)amino)-1-methoxy-2-methyl-3-oxopropyl)pyrrolidin-1-yl)-3-methoxy-5-methyl-1-oxoheptan-4-yl)-2-((S)-2-(dimethylamino)-3-methylbutanamido)-N-methylbutanamide

To a stirred room temperature solution of Dov-Abu(3-N₃)-Dil-Dap-Phe-OHformic acid salt (24.0 mg, 0.029 mmol), tert-butyl amine hydrochloride(7.9 mg, 0.072 mmol), HATU (24.5 mg, 0.064 mmol) in DMF (0.2 mL) wasadded Hunig's base (0.022 mL, 0.124 mmol). After 18 h, analysis by LCMSshowed the reaction was complete. The crude reaction mixture waspurified by preparatory RP-HPLC with a Phenomenex Gemini-NX 10μ C-18 110Å column (150×30 mm) using 5% to 95% MeCN in 0.1% aqueous ammoniumhydroxide as the eluent. A total of 15.4 mg of the title compound wasobtained (0.017 mmol, 59%). LCMS RT=1.27 min (Method B); ESI-MS m/z828.8 [M+H]⁺; HRMS m/z 828.5671 [C₃₉H₆₆N₆O₈+H]⁺.

Example 43 methyl((2R,3R)-3-((S)-1-((3R,4S,5S)-4-((2S,3S)-3-azido-2-((S)-2-(dimethylamino)-3-methylbutanamido)-N-methylbutanamido)-3-methoxy-5-methylheptanoyl)pyrrolidin-2-yl)-3-methoxy-2-methylpropanoyl)-L-valinate

To a stirred 23° C. suspension of Boc-Dap-OH dicyclohexylamine (4.00 g,8.54 mmol) and H-Val-OMe HCl salt (1.80 g, 10.7 mmol) in CH₂Cl₂ (20 mL)was added DIEA (4.44 g, 6.00 mL, 34.4 mmol), followed by the addition ofHATU (2.15 g, 2.00 mL, 0.013 mol). After 10 h, analysis by LCMS showedthe reaction was complete. The volatile organics were evaporated invacuo to give crude product, that was used without further purification.A total of 6.80 g of Boc-Dap-Val-OMe was obtained as a colorless oil(17.0 mmol, 80%). LCMS RT=1.33 min (Method B); ESI-MS m/z 401.6 [M+H]⁺.

A 23° C. suspension of Boc-Dap-Val-OMe (6.80 g, 17.0 mmol) in 4.0 M HClin dioxane (20 mL) was stirred. After 4 h, analysis by LCMS showed thereaction was complete. The volatile organics were evaporated in vacuo togive crude product that was used without further purification. A totalof 4.57 g of H-Dap-Val-OMe was obtained as the HCl salt (13.6 mmol,80%). LCMS RT=0.726 min (Method B); ESI-MS m/z 301.45 [M+H]⁺.

To a stirred 23° C. suspension of Dov-Abu(3-N₃)-Dil-OH TFA salt (0.144g, 0.253 mmol) and H-Dap-Val-OMe HCl salt (0.332 g, 0.985 mmol) in DMF(10 mL) was added DIEA (0.222 g, 0.300 mL, 1.72 mmol), followed by theaddition of HATU (0.240 g, 0.631 mmol). After 10 h, analysis by LCMSshowed the reaction was complete. The crude reaction was diluted withsaturated sodium bicarbonate (10 mL) and extracted with EtOAc (20 mL×3).The combined organic fractions were washed with brine, dried over a padof magnesium sulfate, filtered, and concentrated in vacuo. The crude oilwas purified by preparatory RP-HPLC with a Phenomenex Gemini-NX 10μ C-18110 Å column (150×30 mm) using 5% to 90% MeCN in 0.1% aqueous NH₄OH asthe eluent. A total of 129 mg of the title compound was obtained as apale yellow solid (0.175 mmol, 55%). LCMS RT=1.15 min (Method B); ESI-MSm/z 739.75 [M+H]⁺; HRMS m/z 739.5074 [C₃₆H₆₆N₈O₈+H]⁺.

Example 44 methyl((2R,3R)-3-((S)-1-((3R,4S,5S)-4-((S)-6-amino-2-((S)-2-(dimethylamino)-3-methylbutanamido)-N-methylhexanamido)-3-methoxy-5-methylheptanoyl)pyrrolidin-2-yl)-3-methoxy-2-methylpropanoyl)-L-phenylalaninate

To a stirred 23° C. suspension of Boc-Lys(Fmoc)-OH (5.60 g, 12.0 mmol)and H-Dil-OtBu HCl (3.06 g, 10.9 mmol) in EtOAc (20 mL) was added DIEA(4.44 g, 6.00 mL, 34.4 mmol). The solution was cooled to 0° C. andstirred for 0.5 h. After 0.5 h, additional DIEA (4.44 g, 6.00 mL, 34.4mmol) was added to the reaction mixture and the 0° C. reaction wasstirred for 0.5 h. Then CMPI (4.20 g, 16.4 mmol) was added to thereaction mixture which was allowed to slowly warm to room temperatureand stirred for 10 h. The crude reaction was washed with 1 M HCl (30mL×2), followed by brine (25 mL×2). The organic fraction was dried overa pad of magnesium sulfate, filtered, and concentrated in vacuo. A totalof 7.38 g of Boc-Lys(Fmoc)-Dil-OtBu was obtained as a pale yellow solid(10.4 mmol, 86%). LCMS RT=1.85 min (Method B); ESI-MS m/z 710.1 [M+H]⁺.

A 23° C. suspension of Boc-Lys(Fmoc)-Dil-OtBu (7.38 g, 10.4 mmol) in 4.0M HCl in dioxane (10.0 mL) was stirred. After 10 h, analysis by LCMSshowed the reaction was complete. The volatile organics were evaporatedin vacuo to give crude product that was used without furtherpurification. A total of 7.05 g of H-Lys(Fmoc)-Dil-OH was obtained as apale yellow HCl salt (0.012 mol, 89%). LCMS RT=1.20 min (Method B);ESI-MS m/z 554.54 [M+H]⁺.

To a stirred 23° C. suspension of Dov-OH (2.50 g, 17.2 mmol) in DMF (20mL) was added DIEA (4.44 g, 6.00 mL, 0.034 mol), followed by theaddition of HATU (4.82 g, 12.6 mmol). After 5 min, H-Lys(Fmoc)-Dil-OHHCl salt (7.05 g, 0.012 mol) was added to the reaction mixture. After 4h, analysis by LCMS showed the reaction was complete. The crude reactionmixture was diluted with saturated sodium bicarbonate (10 mL) andextracted with EtOAc (20 mL×2). The combined organic fractions werewashed with brine, dried over a pad of magnesium sulfate, filtered, andconcentrated in vacuo. The crude oil was purified by preparatory RP-HPLCwith a Phenomenex Gemini NX-C18 10μ 110 Å column (150×30 mm) using 5% to95% MeCN in 0.1% aqueous NH₄OH as the eluent. A total of 4.45 g ofDov-Lys(Fmoc)-Dil-OH was obtained as a pale yellow solid (6.54 mmol,78%). LCMS RT=1.21 min (Method B); ESI-MS m/z 681.68 [M+H]⁺.

To a stirred 23° C. suspension of Dov-Lys(Fmoc)-Dil-OH (2.17 g, 3.19mmol) and H-Dap-OMe TFA salt (1.78 g, 3.85 mmol) in DMF (10 mL) wasadded DIEA (1.65 g, 2.20 mL, 12.7 mmol), followed by the addition ofHATU (1.94 g, 5.10 mmol). After 10 h, analysis by LCMS showed thereaction was complete. The crude reaction was diluted with saturatedsodium bicarbonate (10 mL) and extracted with EtOAc (20 mL×3). Thecombined organic fractions were washed with brine, dried over a pad ofmagnesium sulfate, filtered, and concentrated in vacuo. The crude oilwas purified by preparatory RP-HPLC with a Phenomenex Gemini 10 Max-RP110 Å column (150×30 mm) using 5% to 90% MeCN in 0.1% aqueous NH₄OH asthe eluent. A total of 2.25 g of Dov-Lys(Fmoc)-Dil-Dap-Phe-OMe wasobtained as a pale yellow solid (2.23 mmol, 70%). LCMS RT=1.29 min(Method B); ESI-MS m/z 1011.77 [M+H]⁺.

To a stirred 23° C. suspension of Dov-Lys(Fmoc)-Dil-Dap-Phe-OMe (2.25 g,2.23 mmol) in acetonitrile (20 mL) was added piperidine (5 mL). After 2h, analysis by LCMS showed the reaction was complete. To the crudereaction solution was added hexanes. The acetonitrile layer wasconcentrated in vacuo. The crude oil was purified by preparatory RP-HPLCwith a Phenomenex Gemini NX-C18 10μ 110 Å column (150×30 mm) using 5% to95% MeCN in 0.1% aqueous NH₄OH as the eluent. A total of 898 mg of thetitle compound was obtained as a white solid (1.14 mmol, 51%). LCMSRT=0.840 min (Method B); ESI-MS m/z 789.5 [M+H]⁺; HRMS m/z 789.5482[C₄₂H₇₂N₆O₈+H]⁺.

Example 45 methyl((2R,3R)-3-((S)-1-((3R,4S,5S)-4-((2S,4S)-4-azido-1-(dimethyl-L-valyl)-N-methylpyrrolidine-2-carboxamido)-3-methoxy-5-methylheptanoyl)pyrrolidin-2-yl)-3-methoxy-2-methylpropanoyl)-L-phenylalaninate

To a stirred 23° C. suspension of cis-Fmoc-Pro(4-N₃)—OH (2.00 g, 5.27mmol) and H-Dil-OtBu HCl (1.49 g, 5.27 mmol) in EtOAc (20 mL) was addedDIEA (2.22 g, 3.0 mL, 17.2 mmol). The solution was cooled to 0° C. andstirred for 0.5 h. To the cooled reaction mixture was added additionalDIEA (2.22 g, 3.0 mL, 17.2 mmol) and the 0° C. reaction mixture wasstirred for 0.5 h. Then CMPI (2.03 g, 7.93 mmol) was added to thereaction mixture which was allowed to slowly warm to room temperatureand stirred for 10 h. The crude reaction was washed with 1 M HCl (30mL×2), followed by brine (25 mL×2). The organic fraction was dried overa pad of magnesium sulfate, filtered, and concentrated in vacuo. Thecrude product was used without further purification. A total of 3.13 gof cis-Fmoc-Pro(4-N₃)-Dil-OtBu was obtained as a yellow oil (5.05 mmol,76%). LCMS RT=1.73 min (Method B); ESI-MS m/z 621.46 [M+H]⁺.

To a stirred 23° C. solution of cis-Fmoc-Pro(4-N₃)-Dil-OtBu (3.13 g,4.04 mmol) in acetonitrile (20 mL) was added piperidine (10 mL). After10 h, analysis by LCMS showed the reaction was complete. To the crudereaction mixture was added hexanes. The acetonitrile layer wasconcentrated in vacuo and the crude product was used without furtherpurification. A total of 1.57 g of H-Pro(4-N₃)-Dil-OtBu was obtained asa clear oil (3.95 mmol, 88%). LCMS RT=1.19 min (Method B); ESI-MS m/z398.50 [M+H]⁺.

To a stirred 23° C. suspension of Dov-OH (1.03 g, 7.12 mmol) andH-Pro(4-N₃)-Dil-OtBu (1.57 g, 3.56 mmol) in DMF (10 mL) was added DIEA(1.84 g, 2.50 mL, 0.014 mol), followed by the addition of HATU (2.03 g,5.33 mmol). After 3 h, analysis by LCMS showed the reaction wascomplete. The crude reaction was diluted with saturated sodiumbicarbonate (10 mL) and extracted with EtOAc (20 mL×2). The combinedorganic fractions were washed with brine, dried over a pad of magnesiumsulfate, filtered, and concentrated in vacuo. The crude oil was purifiedby preparatory RP-HPLC with a Phenomenex Gemini NX-C18 10μ 110 Å column(150×30 mm) using 5% to 95% MeCN in 0.1% aqueous NH₄OH as the eluent. Atotal of 817 mg of Dov-Pro(4-N₃)-Dil-OtBu was obtained as a white solid(1.56 mmol, 44%). LCMS RT=1.21 min (Method B); ESI-MS m/z 525.28 [M+H]⁺.

A 23° C. suspension of Dov-Pro(4-N₃)-Dil-OtBu (0.817 g, 1.56 mmol) in3.0 M HCl dioxane was stirred. After 10 h, analysis by LCMS showed thereaction was complete. The volatile organics were evaporated in vacuo togive crude product that was used without further purification. A totalof 704 mg of Dov-Pro(4-N₃)-Dil-OH was obtained as the HCl salt (1.39mmol, 89%). LCMS RT=0.676 min (Method B); ESI-MS m/z 469.44 [M+H]⁺.

To a stirred 23° C. suspension of Dov-Pro(4-N₃)-Dil-OH HCl salt (0.120g, 0.256 mmol) and H-Dap-Phe-OMe TFA salt (0.143 g, 0.310 mmol) in DMF(10 mL) was added DIEA (0.132 g, 0.200 mL, 1.02 mmol), followed by theaddition of HATU (0.156 g, 0.410 mmol). After 10 h, analysis by LCMSshowed the reaction was complete. The crude reaction was diluted withsaturated sodium bicarbonate (10 mL) and extracted with EtOAc (20 mL×3).The combined organic fractions were washed with brine, dried over a padof magnesium sulfate, filtered, and concentrated in vacuo. The crude oilwas purified by preparatory RP-HPLC with a Phenomenex Gemini NX-C18 10μ110 Å column (150×30 mm) using 5% to 90% MeCN in 0.1% aqueous NH₄OH asthe eluent. A total of 37.0 mg of the title compound was obtained as ayellow solid (0.046 mmol, 18%) was obtained as a yellow solid. LCMSRT=1.13 min (Method B); ESI-MS m/z 799.43 [M+H]⁺; HRMS m/z 799.5064[C₄₁H₆₆N₈O₈+H]⁺.

Example 46(S)-3-((S)-2-(dimethylamino)-3-methylbutanamido)-4-(((3R,4S,5S)-3-methoxy-1-((S)-2-((1R,2R)-1-methoxy-3-(((S)-1-methoxy-1-oxo-3-phenylpropan-2-yl)amino)-2-methyl-3-oxopropyl)pyrrolidin-1-yl)-5-methyl-1-oxoheptan-4-yl)(methyl)amino)-4-oxobutanoicAcid

To a stirred 23° C. suspension of Boc-Asp(OBzl)-OH (5.00 g, 15.5 mmol)and H-Dil-OtBu TFA salt (4.36 g, 15.5 mmol) in EtOAc (20 mL) was addedDIEA (6.00 g, 8.10 mL, 46.41 mmol). The reaction mixture was cooled to0° C. and stirred for 0.5 h. After 0.5 h, additional DIEA (6.00 g, 8.01mL, 46.4 mmol) was added to the reaction mixture and stirred for 0.5 h.Then CMPI (5.93 g, 23.2 mmol) was added to the reaction mixture whichwas allowed to slowly warm to room temperature and stirred for 10h. Thecrude reaction was washed with 1 M HCl (30 mL×2), followed by brine (25mL×2). The organic fraction was dried over anhydrous magnesium sulfate,filtered, and concentrated in vacuo. The crude product was used withoutfurther purification. A total of 7.85 g of Boc-Asp(OBzl)-Dil-OtBu wasobtained as a brown oil (13.9 mmol, 90%). LCMS RT=1.72 min (Method B);ESI-MS m/z 565.3 [M+H]⁺.

A 23° C. solution of Boc-Asp(OBzl)-Dil-OtBu (7.85 g, 13.9 mmol) in 4.0 MHCl in dioxane (20 mL) was stirred. After 10 h, analysis by LCMS showedthe reaction was complete. The volatile organics were evaporated invacuo to give crude product that was used without further purification.A total of 7.85 g of enriched H-Asp(OBzl)-Dil-OH was obtained as the HClsalt. LCMS RT=0.951 min (Method B); ESI-MS m/z 409.40 [M+H]⁺.

To a stirred 23° C. suspension of Dov-OH (3.00 g, 20.7 mmol) in DMF (20mL) was added DIEA (6.96 g, 9.40 mL, 0.054 mol) and HATU (7.60 g, 19.9mmol), followed by the addition of enriched H-Asp(OBzl)-Dil-OH HCl salt(7.85 g). After 4 h, analysis by LCMS showed the reaction was complete.The crude reaction was diluted with saturated sodium bicarbonate (10 mL)and extracted with EtOAc (20 mL×2). The combined organic fractions werewashed with brine, dried over a pad of magnesium sulfate, filtered, andconcentrated in vacuo. The crude oil was purified by preparatory RP-HPLCwith a Phenomenex Gemini NX-C18 10μ 110 Å column (150×30 mm) using 5% to95% MeCN in 0.1% aqueous NH₄OH as the eluent. A total of 2.00 g ofDov-Asp(OBzl)-Dil-OH was obtained as a white solid (3.74 mmol, 28%).LCMS RT=1.10 min (Method B); ESI-MS m/z 536.5 [M+H]⁺.

To a stirred 23° C. suspension of Dov-Asp(OBzl)-Dil-OH (1.00 g, 1.87mmol) and H-Dap-Phe-OMe TFA salt (1.04 g, 2.25 mmol) in DMF (10 mL) wasadded DIEA (0.970 g, 1.30 mL, 7.47 mmol), followed by the addition ofHATU (1.14 g, 2.99 mmol). After 10 h, analysis by LCMS showed thereaction was complete. The crude reaction was diluted with saturatedsodium bicarbonate (10 mL) and extracted with EtOAc (20 mL×3). Thecombined organic fractions were washed with brine, dried over a pad ofmagnesium sulfate, filtered, and concentrated in vacuo. The crude oilwas purified by preparatory RP-HPLC with a Phenomenex Gemini NX-C18 10μ110 Å column (150×30 mm) using 5% to 90% MeCN in 0.1% aqueous NH₄OH asthe eluent. A total of 1.25 g of Dov-Asp(OBzl)-Dil-Dap-Phe-OMe (1.44mmol, 77%) was obtained as an orange solid. LCMS RT=1.21 min (Method B);ESI-MS m/z 866.6 [M+H]⁺.

To a stirred 23° C. suspension of Dov-Asp(OBzl)-Dil-Dap-Phe-OMe (0.525g, 0.606 mmol) in MeOH (10 mL) was added 10% Pd/C (50 mg), followed bythe addition of a hydrogen atmosphere. After 5 h, analysis by LCMSshowed the reaction was complete. The crude reaction mixture wasfiltered over a pad of diatomaceous earth, followed by evaporation ofthe volatile organics. The crude oil was purified by preparatory RP-HPLCwith a Phenomenex Gemini NX-C18 10μ 110 Å column (150×30 mm) using 5% to90% MeCN in 0.1% aqueous NH₄OH as the eluent. A total of 6.00 mg of thetitle compound was obtained as a white solid (0.008 mmol, 1%). LCMSRT=1.04 min (Method B); ESI-MS m/z 776.43 [M+H]⁺; HRMS m/z 776.4794[C₄₀H₆₅N₅O₁₀+H]⁺.

Example 47(2S,3R)-2-((S)-2-(dimethylamino)-3-methylbutanamido)-3-hydroxy-N-((3R,4S,5S)-1-((S)-2-((1R,2R)-3-(((1S,2R)-1-hydroxy-1-phenylpropan-2-yl)amino)-1-methoxy-2-methyl-3-oxopropyl)pyrrolidin-1-yl)-3-methoxy-5-methyl-1-oxoheptan-4-yl)-N-methylbutanamide

To a stirred 23° C. suspension of Dov-Thr(Bzl)-Dil-OH TFA salt (561 mg,0.883 mmol) and H-Dap-(1R,2S)-(−)-Norephedrine TFA salt (345 mg, 0.794mmol) in DMF (20 mL) was added DIEA (417 mg, 562 μL, 3.23 mmol) followedby the addition of HATU (820 mg, 2.15 mmol). After 8 h, analysis by LCMSshowed the reaction was complete. The crude reaction was diluted withsaturated sodium bicarbonate (10 mL) and extracted with EtOAc (20 mL×2).The combined organic fractions were washed with brine, dried over a padof magnesium sulfate, filtered, and concentrated in vacuo. The crude oilwas purified by preparatory RP-HPLC with a Phenomenex Gemini NX-C18 10μ110 Å column (150×30 mm) using 5% to 95% MeCN in 0.1% aqueous NH₄OH asthe eluent. A total of 326 mg ofDov-Thr(Bzl)-Dil-Dap-(1R,2S)-(−)-Norephedrine was obtained as a whitesolid (0.396 mmol, 37%). LCMS RT=1.20 min (Method B); ESI-MS m/z 824.76[M+H]⁺.

To a stirred 23° C. suspension ofDov-Thr(Bzl)-Dil-Dap-(1R,2S)-(−)-Norephedrine (326 mg, 0.396 mmol) inMeOH (10 mL) was loaded onto a continuous flow hydrogenation reactorusing a RaNi (CatCart) and elevated temperature (120° C.) and pressure(80 bar). After elution, analysis by LCMS showed the reaction wascomplete. The volatile organics were evaporated in vacuo to give crudeproduct, that was purified by preparatory RP-HPLC with a PhenomenexGemini NX-C18 10μ 110 Å column (150×30 mm) using 5% to 95% MeCN in 0.1%aqueous NH4OH as the eluent. A total of 15 mg of the title compound wasobtained as a white solid (0.020 mmol, 5%). LCMS RT=1.17 min (Method B);ESI-MS m/z 734.5 [M+H]⁺; HRMS m/z 734.5053 [C₃₉H₆₇N₅O₈+H]⁺.

Example 48 methyl((2R,3R)-3-((S)-1-((3R,4S,5S)-4-((S)-2-((S)-2-(dimethylamino)-3-methylbutanamido)-N,3-dimethylbutanamido)-3-methoxy-5-methylheptanoyl)pyrrolidin-2-yl)-3-methoxy-2-methylpropanoyl)-L-serinate

To a stirred 23° C. suspension of Dov-Val-Dil-OH TFA salt (583 mg, 1.07mmol) and H-Dap-Ser-OMe TFA salt (783 mg, 1.97 mmol) in DMF (10 mL) wasadded DIEA (223 mg, 0.324 mL, 1.72 mmol), followed by the addition ofHATU (1.03 g, 2.71 mmol). After 10 h, analysis by LCMS showed thereaction was complete. The crude reaction was diluted with saturatedsodium bicarbonate (10 mL) and extracted with EtOAc (20 mL×3). Thecombined organic fractions were washed with brine, dried over a pad ofmagnesium sulfate, filtered, and concentrated in vacuo. The crude oilwas purified by preparatory RP-HPLC with a Phenomenex Gemini NX-C18 10μ110 Å column (150×30 mm) using 5% to 90% MeCN in 0.1% aqueous NH₄OH asthe eluent. A total of 704 mg of the title compound was obtained as apale yellow solid (1.01 mmol, 74%). LCMS RT=0.917 min (Method B); ESI-MSm/z 700.43 [M+H]⁺; HRMS m/z 700.4843 [C₃₅H₆₅N₅O₉+H]⁺.

Example 49 methyl((2R,3R)-3-((S)-1-((3R,4S,5S)-4-((2S,3S)-3-azido-2-((S)-2-(dimethylamino)-3-methylbutanamido)-N-methylbutanamido)-3-methoxy-5-methylheptanoyl)pyrrolidin-2-yl)-3-methoxy-2-methylpropanoyl)-L-isoleucinate

To a stirred 23° C. suspension of Boc-Dap-OH dicyclohexylamine salt(6.62 g, 14.1 mmol) and H-Ile-OMe (3.08 g, 21.2 mmol) in CH₂Cl₂ (20 mL)was added DIEA (7.30 g, 9.90 mL, 56.5 mmol), followed by the addition ofHATU (3.44 g, 3.20 mL, 0.021 mol). After 10 h, analysis by LCMS showedthe reaction was complete. The volatile organics were evaporated invacuo to give crude product that was used without further purification.A total of 8.56 g of Boc-Dap-Ile-OMe was obtained as a brown oil (20.7mmol, 88%). LCMS RT=1.51 min (Method B); ESI-MS m/z 415.16 [M+H]⁺.

To a stirred 23° C. suspension of Boc-Dap-Ile-OMe (6.62 g, 16.0 mmol) inCH₂Cl₂ (20 mL) was added TFA (10 mL). After 10 h, analysis by LCMSshowed the reaction was complete. The volatile organics were evaporatedin vacuo. The crude oil was purified by preparatory RP-HPLC with aPhenomenex Gemini NX-C18 10μ 110 Å column (150×30 mm) using 5% to 95%MeCN in 0.1% aqueous NH₄OH as the eluent. A total of 3.55 g ofH-Dap-Ile-OMe was obtained as a yellow solid (8.29 mmol, 52%). LCMSRT=0.691 min (Method B); ESI-MS m/z 315.16 [M+H]⁺.

To a stirred 23° C. suspension of Dov-Abu(3-N₃)-Dil-OH TFA salt (150 mg,0.329 mmol) and H-Dap-Ile-OMe (207 mg, 0.657 mmol) in DMF (10 mL) wasadded DIEA (170 mg, 0.220 mL, 1.31 mmol), followed by the addition ofHATU (251 mg, 0.657 mmol). After 10 h, analysis by LCMS showed thereaction was complete. The crude reaction was diluted with saturatedsodium bicarbonate (10 mL) and extracted with EtOAc (20 mL×3). Thecombined organic fractions were washed with brine, dried over a pad ofmagnesium sulfate, filtered, and concentrated in vacuo. The crude oilwas purified by preparatory RP-HPLC with a Phenomenex Gemini NX-C18 10μ110 Å column (150×30 mm) using 5% to 90% MeCN in 0.1% aqueous NH₄OH asthe eluent. A total of 148 mg of the title compound was obtained as apale yellow solid (0.197 mmol, 60%). LCMS RT=1.43 min (Method B); ESI-MSm/z 753.48 [M+H]⁺; HRMS m/z 753.5224 [C₃₇H₆₈N₈O₈+H]⁺.

Example 50(2S,3S)-3-amino-N-((3R,4S,5S)-1-((S)-2-((1R,2R)-3-(((S)-1-amino-1-oxo-3-phenylpropan-2-yl)amino)-1-methoxy-2-methyl-3-oxopropyl)pyrrolidin-1-yl)-3-methoxy-5-methyl-1-oxoheptan-4-yl)-2-((S)-2-(dimethylamino)-3-methylbutanamido)-N-methylbutanamide

To a stirred room temperature solution of Dov-Abu(3-N₃)-Dil-Dap-Phe-NH₂(13.4 mg, 0.017 mmol) in THF (0.1 mL) was added trimethylphosphine inTHF (1 M, 0.035 mL, 0.035 mmol). After 3 h, additionaltrimethylphosphine was added (1 M, 0.020 mL, 0.020 mmol). After another1 h, analysis by LCMS showed that the reaction was complete. Thesolution was diluted with water and DMF and allowed to stand for 30 min.The crude reaction mixture was purified by preparatory RP-HPLC with aPhenomenex Gemini-NX 10μ C18 110 Å column (150×30 mm) using 5% to 95%MeCN in 0.1% aqueous ammonium hydroxide as the eluent. A total of 5.8 mgof the title compound was obtained (0.008 mmol, 45%). LCMS RT=0.85 min(Method B); ESI-MS m/z 746.6 [M+H]⁺; HRMS m/z 746.5173 [C₃₉H₆₇N₇₀₇+H]⁺.

Example 51(2S,3S)-3-amino-N-((3R,4S,5S)-1-((S)-2-((1R,2R)-3-(((S)-1-(tert-butylamino)-1-oxo-3-phenylpropan-2-yl)amino)-1-methoxy-2-methyl-3-oxopropyl)pyrrolidin-1-yl)-3-methoxy-5-methyl-1-oxoheptan-4-yl)-2-((S)-2-(dimethylamino)-3-methylbutanamido)-N-methylbutanamide

To a stirred room temperature solution ofDov-Abu(3-N₃)-Dil-Dap-Phe-NHt-Bu (15.4 mg, 0.019 mmol) in THF (0.1 mL)was added trimethylphosphine in THF (1 M, 0.037 mL, 0.037 mmol). After 3h, additional trimethyl phosphine was added (1 M, 0.020 mL, 0.020 mmol)was added. After another 1 h, analysis by LCMS showed that the reactionwas complete. The reaction solution was diluted with water and DMF andallowed to stand for 30 min. The crude reaction mixture was purified bypreparatory RP-HPLC with a Phenomenex Gemini-NX 10μ C -18 110 Å column(150×30 mm) using 5% to 95% MeCN in 0.1% aqueous ammonium hydroxide asthe eluent. A total of 2.7 mg of the title compound was obtained (0.003mmol, 18%). LCMS RT=0.85 min (Method B); ESI-MS m/z 746.6 [M+H]⁺; HRMSm/z 802.5799 [C₄₃H₇₅N₇O₇+H]⁺.

Example 52 methyl((2R,3R)-3-((S)-1-((3R,4S,5S)-4-((S)-3-azido-N-methyl-2-((S)-3-methyl-2-(methylamino)butanamido)propanamido)-3-methoxy-5-methylheptanoyl)pyrrolidin-2-yl)-3-methoxy-2-methylpropanoyl)-L-phenylalaninate

To a stirred 23° C. suspension of Fmoc-MeVal-OH (1.03 g, 2.90 mmol) inDMF (10 mL) was added DIEA (1.35 mL, 7.74 mmol), followed by theaddition of HATU (1.11 g, 2.90 mmol). After 5 min H-4-Azido-Ala-Dil-OH(0.610 g, 2.90 mmol) was added to the reaction mixture. After 10 h,analysis by LCMS showed that the reaction was complete. The crudereaction was diluted with saturated sodium bicarbonate (15 mL) andextracted with EtOAc (40 mL×2). The combined organic fractions werewashed with brine, dried over a pad of magnesium sulfate, filtered, andconcentrated in vacuo. The crude oil was purified by preparatory RP-HPLCwith a Phenomenex Gemini NX-C18 10μ 110 Å column (150×30 mm) using 5% to95% MeCN in 0.1% aqueous NH₄OH as the eluent. A total of 348 mg ofFmoc-MeVal -4-Azido-Ala-Dil-OH (0.535 mmol, 28%) was obtained as ayellow oil. LCMS RT=1.80 min (Method B); ESI-MS m/z 651.3 [M+H]⁺.

To a stirred 23° C. suspension of Fmoc-MeVal-4-Azido-Ala-Dil-OH (348.00mg, 0.535 mmol) and H-Dap-Phe-OMe (372.66 mg, 1.07 mmol) in DMF (10 mL)was added DIEA (276 mg, 0.400 mL, 2.14 mmol) followed by the addition ofHATU (408 mg, 1.07 mmol). After 10 h, analysis by LCMS showed thereaction was complete with deprotection of the Fmoc group occurringconcurrently. The crude reaction was diluted with saturated sodiumbicarbonate (10 mL) and extracted with EtOAc (20 mL×3). The combinedorganic fractions were washed with brine, dried over a pad of magnesiumsulfate, filtered, and concentrated in vacuo. The crude oil was purifiedby preparatory RP-HPLC with a Phenomenex Gemini NX-C18 10μ 110 Å column(150×30 mm) using 10% to 90% MeCN in 0.1% aqueous formic acid as theeluent. A total of 5.00 mg of the title compound was obtained (0.006mmol, 1%). LCMS RT=1.08 min (Method B); ESI-MS m/z 759.5 [M+H]⁺; HRMSm/z 759.4753 [C₃₈H₆₂N₈O₈+H]⁺.

Example 53 methyl((2R,3R)-3-((S)-1-((3R,4S,5S)-4-((2S,3S)-3-azido-N-methyl-2-((S)-3-methyl-2-(methylamino)butanamido)butanamido)-3-methoxy-5-methylheptanoyl)pyrrolidin-2-yl)-3-methoxy-2-methylpropanoyl)-L-phenylalaninate

To a stirred 23° C. suspension of Fmoc-MeVal-OH (2.46 g, 6.96 mmol) andH-Abu(3-N₃)-Dil-OtBu (2.46 g, 6.38 mmol) in DMF (10 mL) was added DIEA(3.30 g, 4.5 mL, 25.5 mmol), followed by the addition of HATU (3.65 g,9.57 mmol). After 6 h, analysis by LCMS showed the reaction wascomplete. The crude reaction mixture was purified by preparatory RP-HPLCwith a Phenomenex Gemini NX-C18 10μ 110 Å column (150×30 mm) using 5% to95% MeCN in 0.1% aqueous formic acid as the eluent. A total of 3.16 g ofFmoc-MeVal-Abu(3-N₃)-Dil-OtBu (4.12 mmol, 65%) was obtained as theformic acid salt. LCMS RT=2.08 min (Method A); ESI-MS m/z 722.7 [M+H]⁺.

To a stirred 23° C. suspension of Fmoc-MeVal-Abu(3-N₃)-Dil-OtBu formicacid salt (3.16 g, 4.12 mmol) in CH₂Cl₁₂ (5.0 mL) was added TFA (10.0mL). After 14 h, analysis by LCMS showed the reaction was complete. Thevolatile organics were evaporated in vacuo to give an oil. The crude oilwas purified by preparatory RP-HPLC with a Phenomenex Gemini NX-C18 10μ110 Å column (150×30 mm) using 5% to 95% MeCN in 0.1% aqueous TFA as theeluent. A total of 2.26 g of Fmoc-MeVal-Abu(3-N₃)-Dil-OH (2.90 mmol,66%) was obtained as the TFA salt. LCMS RT=1.80 min (Method B); ESI-MSm/z 665.3 [M+H]⁺.

To a stirred 23° C. suspension of Fmoc-MeVal-Abu(3-N₃)-Dil-OH TFA salt(80.0 mg, 0.103 mmol) and H-Dap-Phe-OMe TFA salt (83.9 mg, 0.182 mmol)in DMF (2 mL) was added DIEA (0.083 mL, 0.481 mmol) followed by theaddition of HATU (91.8 mg, 0.241 mmol). After 12 h, analysis by LCMSshowed the reaction was complete. The crude reaction mixture was dilutedwith DMF and purified by preparatory RP-HPLC with a Phenomenex GeminiNX-C18 10μ 110 Å column (150×30 mm) using 10% to 90% MeCN in 0.1%aqueous formic acid as the eluent. A total of 97.0 mg ofFmoc-MeVal-Abu(3-N₃)-Dil-Dap-Phe-OMe (0.093 mmol, 77%) was obtained asthe formic acid salt. LCMS RT=1.90 min (Method B); ESI-MS m/z 995.6[M+H]⁺.

To a stirred 23° C. solution of Fmoc-MeVal-Abu(3-N₃)-Dil-Dap-Phe-OMe(97.0 mg, 0.093 mmol) in acetonitrile (10 mL) was added piperidine (5mL). After 5 h, analysis by LCMS showed the reaction was complete. Tothe crude reaction solution was added hexanes (25 mL×3) in order toextract non-polar by-products. The acetonitrile layer was concentratedin vacuo and the crude oil was purified by preparatory RP-HPLC with aPhenomenex Gemini NX-C18 110 Å column (150×30 mm) using 5% to 95% MeCNin 0.1% aqueous NH₄OH as the eluent. A total of 74.0 mg of the titlecompound (0.096 mmol, 103%) was obtained as a white solid. LCMS RT=1.09min (Method B); ESI-MS m/z 773.5 [M+H]⁺; HRMS m/z 773.4911[C₃₉H₆₄N₈O₈+H]⁺.

Example 54(2S,3S)—N-((3R,4S,5S)-1-((S)-2-((1R,2R)-3-(((S)-1-amino-1-oxo-3-phenylpropan-2-yl)amino)-1-methoxy-2-methyl-3-oxopropyl)pyrrolidin-1-yl)-3-methoxy-5-methyl-1-oxoheptan-4-yl)-3-azido-N-methyl-2-((S)-3-methyl-2-(methylamino)butanamido)butanamide

To a stirred 23° C. suspension of Bod-Dap-OH dicyclohexylamine (10.0 g,21.3 mmol) and H-Phe-NH₂ HCl salt (6.42 g, 32.0 mmol) in CH₂Cl₂ (20.0mL) was added DIEA (11.0 g, 14.9 mL, 85.3 mmol) followed by the additionof DEPC (5.19 g, 4.80 mL, 0.032 mol). After 10 h, analysis by LCMSshowed the reaction was complete. The crude reaction was washed with H₂O(25 mL×2), followed by brine (25 mL×2). The organic fraction was driedover a pad of magnesium sulfate, filtered and concentrated in vacuo. Thecrude orange oil was purified by flash chromatography (silica gel 40 μm,60 Å, size) using 2% to 10% methanol in CH₂Cl₂ as the eluent. A total of7.25 g of Boc-Dap-Phe-NH₂ (16.7 mmol, 78%) was obtained as a yellow oil.LCMS RT=1.28 min (Method B); ESI-MS m/z 434.19 [M+H]⁺.

To a stirred 23° C. suspension of Boc-Dap-Phe-NH₂ (7.25 g, 16.7 mmol) inCH₂Cl₂ (10 mL) was added TFA (10 mL). After 5 h, analysis by LCMS showedthe reaction was complete. The volatile organics were evaporated invacuo to give crude product, which was used without furtherpurification. A total of 6.00 g of H-Dap-Phe-NH₂ was obtained as anorange solid (13.4 mmol, 80%). LCMS RT=0.691 min (Method B); ESI-MS m/z334.17 [M+H]⁺.

To a stirred 23° C. suspension of Fmoc-MeVal-Abu(3-N₃)-Dil-OH TFA salt(456 mg, 0.586 mmol) and H-Dap-Phe-NH₂ TFA salt (457 mg, 1.02 mmol) inDMF (10 mL) was added DIEA (0.350 g, 0.500 mL, 2.74 mmol) followed bythe addition of HATU (0.520 g, 1.37 mmol). After 10 h, analysis by LCMSshowed the reaction was complete. The crude reaction was purified bypreparatory RP-HPLC with a Phenomenex Gemini NX-C18 10μ 110 Å column(150×30 mm) using 10% to 90% MeCN in 0.1% aqueous formic acid as theeluent. A total of 526 mg of Fmoc-MeVal-Abu(3-N₃)-Dil-Dap-Phe-NH₂ wasobtained as the formic acid salt (0.513 mmol, 75%). LCMS RT=1.81 min(Method B); ESI-MS m/z 980.39 [M+H]⁺.

To a stirred 23° C. solution of Fmoc-MeVal-Abu(3-N₃)-Dil-Dap-Phe-NH₂(525 mg, 0.513 mmol) in acetonitrile (10 mL) was added piperidine (5mL). After 2 h, analysis by LCMS showed the reaction was complete. Tothe crude reaction solution was added hexanes (15 mL×3). Theacetonitrile layer was concentrated in vacuo. The crude oil was purifiedby preparatory RP-HPLC with a Phenomenex Gemini NX-C18 10μ 110 Å column(150×30 mm) using 5% to 95% MeCN in 0.1% aqueous TFA as the eluent. Atotal of 354 mg of the title compound was obtained as the TFA salt(0.406 mmol, 79%). LCMS RT=1.15 min (Method B); ESI-MS m/z 758.24[M+H]⁺; HRMS m/z 758.4915 [C₃₈H₆₃N₉O₇+H]⁺.

Example 55((2S,3S)-3-azido-N-((3R,4S,5S)-1-((S)-2-((1R,2R)-3-(((S)-1-(tert-butylamino)-1-oxo-3-phenylpropan-2-yl)amino)-1-methoxy-2-methyl-3-oxopropyl)pyrrolidin-1-yl)-3-methoxy-5-methyl-1-oxoheptan-4-yl)-N-methyl-2-((S)-3-methyl-2-(methylamino)butanamido)butanamide

To a stirred room temperature solution ofFmoc-MeVal-Abu(3-N₃)-Dil-Dap-Phe-OH (111 mg, 0.113 mmol), tert-butylamine hydrochloride (31.5 mg, 0.287 mmol), HATU (92.0 mg, 0.242 mmol) inDMF (1.0 mL) was added Hunig's base (0.079 mL, 0.454 mmol). After 1 h,analysis by LCMS showed the reaction was complete. The crude reactionmixture was diluted with ethyl acetate and the organic fraction werewashed with 1 N HCl and brine. The organic fraction was dried overmagnesium sulfate, filtered and concentrated under reduced pressure. Thecrude yellow oil was dissolved in piperidine (2.0 mL) and acetonitrile(5.0 mL). After 1 h, analysis by LCMS showed the reaction was complete.The acetonitrile layer was extracted with hexanes (2×) and theacetonitrile layer was concentrated under reduced pressure. The crudeoil was purified by preparatory RP-HPLC with a Phenomenex Gemini-NX 10μC-18 110 Å column (150×30 mm) using 5% to 95% MeCN in 0.1% aqueousammonium hydroxide as the eluent. A total of 50.3 mg of the titlecompound was obtained (0.062 mmol, 55%). LCMS RT=1.29 min (Method B);ESI-MS m/z 814.1 [M+H]⁺; HRMS m/z 814.5541 [C₄₂H₇₁N₉O₇+H]⁺.

Example 56 tert-butyl((2R,3R)-3-((S)-1-((3R,4S,5S)-4-((2S,3S)-3-azido-N-methyl-2-((S)-3-methyl-2-(methylamino)butanamido)butanamido)-3-methoxy-5-methylheptanoyl)pyrrolidin-2-yl)-3-methoxy-2-methylpropanoyl)-L-phenylalaninate

To a stirred 25° C. solution of Boc-Dap-OH dicyclohexylamine salt (6.47g, 13.8 mmol) and H-Phe-OtBu (3.91 g, 15.2 mmol) in DCM (20 mL) wasadded DIEA (8.76 mL, 55.2 mmol), followed by the addition of DEPC (3.12mL, 20.7). After 8 h, analysis by LCMS showed the reaction was complete.The volatile organics were evaporated in vacuo to give crude productthat was used without further purification. A total of 5.35 g ofBoc-Dap-Phe-OtBu was obtained (10.9 mmol, 79%). LCMS RT=2.89 min (MethodA); ESI-MS m/z 491.48 [M+H]⁺.

To a stirred room temperature solution of Boc-Dap-Phe-OtBu (5.25 g, 10.7mmol) in CH₂Cl₁₂ (10.0 mL) was added TFA (10.0 mL). After 12 h, analysisby LCMS showed the reaction was complete. The crude oil was purified bypreparatory RP-HPLC with a Phenomenex Synergi 10μ Max-RP 80 Å column(150×30 mm) using 10% to 90% MeCN in 0.05% aqueous TFA as the eluent. Atotal of 2.85 g of H-Dap-Phe-OtBu was obtained as the TFA salt (5.65mmol, 68%). LCMS RT=1.82 min (Method A); ESI-MS m/z 391.02 [M+H]⁺.

To a stirred 23° C. suspension of Fmoc-MeVal-Abu(3-N₃)-Dil-OH TFA salt(410 mg, 0.527 mmol) and H-Dap-Phe-OtBu TFA salt (482 mg, 0.956 mmol) inDMF (10 mL) was added DIEA (319 mg, 430 μL, 2.47 mmol) followed by theaddition of HATU (470 mg, 1.23 mmol). After 10 h, analysis by LCMSshowed the reaction was complete along with 10% of the Fmoc group beingremoved. The crude reaction mixture was purified by preparatory RP-HPLCwith a Phenomenex Gemini NX-C18 10μ 110 Å column (150×30 mm) using 10%to 90% MeCN in 0.1% aqueous formic acid as the eluent. A total of 26.0mg of the title compound was obtained as a white formic acid salt (0.030mmol, 5%). LCMS RT=1.41 min (Method B); ESI-MS m/z 815.33 [M+H]⁺; HRMSm/z 815.5383 [C₄₂H₇₀N₈O₈+H]⁺.

Example 57((2R,3R)-3-((S)-1-((3R,4S,5S)-4-((2S,3S)-3-azido-N-methyl-2-((S)-3-methyl-2-(methylamino)butanamido)butanamido)-3-methoxy-5-methylheptanoyl)pyrrolidin-2-yl)-3-methoxy-2-methylpropanoyl)-L-phenylalanine

To a stirred 23° C. suspension of Fmoc-MeVal-Abu(3-N₃)-Dil-OH TFA salt(410 mg, 0.527 mmol) and H-Dap-Phe-OtBu TFA salt (482 mg, 0.956 mmol) inDMF (10 mL) was added DIEA (319 mg, 430 μL, 2.47 mmol) followed by theaddition of HATU (470 mg, 1.23 mmol). After 10 h, analysis by LCMSshowed the reaction was complete. The crude reaction mixture waspurified by preparatory RP-HPLC with a Phenomenex Gemini NX-C18 10μ 110Å column (150×30 mm) using 10% to 90% MeCN in 0.1% aqueous formic acidas the eluent. A total of 648 mg ofFmoc-MeVal-Abu(3-N₃)-Dil-Dap-Phe-OtBu was obtained as a white formicacid salt (0.598 mmol, 97%). LCMS RT=1.41 min (Method B); ESI-MS m/z1037.41 [M+H]⁺.

To a stirred 23° C. suspension of Fmoc-MeVal-Abu(3-N₃)-Dil-Dap-Phe-OtBuformic acid salt (648 mg, 0.598 mmol) in CH₂Cl₁₂ (5.00 mL) was added TFA(5.00 mL). After 2 h, analysis by LCMS showed the reaction was complete.The volatile organics were evaporated in vacuo to give a brown oil. Thecrude oil was purified by preparatory RP-HPLC with a Phenomenex GeminiNX-C18 10μ 110 Å column (150×30 mm) using 5% to 95% MeCN in 0.1% aqueousTFA as the eluent. A total of 502 mg ofFmoc-MeVal-Abu(3-N₃)-Dil-Dap-Phe-OH was obtained as the TFA salt (0.458mmol, 77%). LCMS RT=1.81 min (Method B); ESI-MS m/z 981.22 [M+H]⁺.

To a stirred 23° C. solution of Fmoc-MeVal-Abu(3-N₃)-Dil-Dap-Phe-OH TFAsalt (392 mg, 0.382 mmol) in acetonitrile (10 mL) was added piperidine(5 mL). After 2 h, analysis by LCMS showed the reaction was complete. Tothe crude reaction solution was added hexanes (15 mL×3). Theacetonitrile layer was concentrated in vacuo. The crude oil was purifiedby preparatory RP-HPLC with a Phenomenex Gemini NX-C18 10μ 110 Å column(150×30 mm) using 5% to 95% MeCN in 0.1% aqueous TFA as the eluent. Atotal of 360 mg of the enriched title compound was obtained as the TFAsalt. LCMS RT=1.19 min (Method B); ESI-MS m/z 759.13 [M+H]⁺; HRMS m/z759.4755 [C₃₈H₆₂NO₈+H]⁺.

Example 58 tert-butyl((2R,3R)-3-((S)-1-((3R,4S,5S)-4-((2S,3S)-3-azido-2-((S)-2-(dimethylamino)-3-methylbutanamido)-N-methylbutanamido)-3-methoxy-5-methylheptanoyl)pyrrolidin-2-yl)-3-methoxy-2-methylpropanoyl)-L-phenylalaninate

To a stirred 23° C. suspension of Dov-Abu(3-N₃)-Dil-OH TFA salt (327 mg,0.574 mmol) and H-Dap-Phe-OtBu TFA salt (0.340 g, 0.675 mmol) in DMF (10mL) was added DIEA (0.370 g, 0.500 mL, 2.87 mmol) followed by theaddition of HATU (0.550 g, 1.43 mmol). After 10 h, analysis by LCMSshowed the reaction was complete. The crude reaction was purified bypreparatory RP-HPLC with a Phenomenex Gemini NX-C18 10μ 110 Å column(150×30 mm) using 10% to 90% MeCN in 0.1% aqueous formic acid as theeluent. A total of 453 mg (0.518 mmol, 72%) of the title compound wasobtained as the formic acid salt. LCMS RT=1.42 min (Method B); ESI-MSm/z 828.94 [M+H]⁺; HRMS m/z 829.5536 [C₄₃H₇₂NO₈+H]⁺.

Example 59(2S,3S)-3-azido-2-((S)-2-(dimethylamino)-3-methylbutanamido)-N-((3R,4S,5S)-3-methoxy-1-((S)-2-((1R,2R)-1-methoxy-2-methyl-3-oxo-3-(((S)-2-phenyl-1-(1H-tetrazol-5-yl)ethyl)amino)propyl)pyrrolidin-1-yl)-5-methyl-1-oxoheptan-4-yl)-N-methylbutanamide

To a stirred 23° C. suspension of Boc-Dap-OH dicyclohexylamine salt(2.48 g, 5.29 mmol) and (S)-2-Phenyl-1-(1H-tetrazol-5-yl)ethanamine(1.00 g, 5.29 mmol) in CH₂Cl₂ (20.0 mL) was added DIEA (2.73 g, 3.7 mL,21.1 mmol) followed by the addition of DEPC (1.29 g, 1.20 mL, 7.93mmol). After 10 h, analysis by LCMS showed the reaction was complete.The volatile organics were evaporated in vacuo. The crude oil waspurified by preparatory RP-HPLC with a Phenomenex Gemini NX-C18 10μ 110Å column (150×30 mm) using 5% to 95% MeCN in 0.1% aqueous formic acid asthe eluent. A total of 2.52 g ofBoc-Dap-(S)-2-Phenyl-1-(1H-tetrazol-5-yl)ethanamine (4.99 mmol, 94%) wasobtained as the formic acid salt. LCMS RT=1.35 min (Method B); ESI-MSm/z 459.2 [M+H]⁺.

To a stirred 23° C. suspension ofBoc-Dap-(S)-2-Phenyl-1-(1H-tetrazol-5-yl)ethanamine (2.52 g, 4.99 mmol)in CH₂Cl₂ (10.0 mL) was added TFA (5.00 mL). After 5 h, analysis by LCMSshowed the reaction was complete. The volatile organics were evaporatedin vacuo to give crude product that was used without furtherpurification. A total of 2.43 g ofH-Dap-(S)-2-Phenyl-1-(1H-tetrazol-5-yl)ethanamine (5.14 mmol, 84%) wasobtained as the TFA salt. LCMS RT=0.575 min (Method B); ESI-MS m/z 359.2[M+H]⁺.

To a stirred 23° C. suspension of Dov-Abu(3-N₃)-Dil-OH TFA salt (305 mg,0.668 mmol) and H-Dap-(S)-2-Phenyl-1-(1H-tetrazol-5-yl)ethanamine TFAsalt (0.36 g, 1.002 mmol) in DMF (10 mL) was added DIEA (0.35 g, 0.5 mL,2.67 mmol) followed by the addition of HATU (509 mg, 1.34 mmol). After10 h, analysis by LCMS showed the reaction was complete. The crudereaction was diluted with saturated sodium bicarbonate (10 mL) andextracted with EtOAc (20 mL×3). The combined organic fractions werewashed with brine, dried over a pad of magnesium sulfate, filtered, andconcentrated in vacuo. The crude oil was purified by preparatory RP-HPLCwith a Phenomenex Gemini NX-C18 10μ 110 Å column (150×30 mm) using 10%to 90% MeCN in 0.1% aqueous formic acid as the eluent. A total of 47.0mg of the title compound was obtained as the formic acid salt (0.056mmol, 8%). LCMS RT=1.24 min (Method B); ESI-MS m/z 797.3 [M+H]⁺; HRMSm/z 797.5139 [C₃₉H₆₄N₁₂O₆+H]⁺.

Example 60(2S,3S)-3-azido-N-((3R,4S,5S)-3-methoxy-1-((S)-2-((1R,2R)-1-methoxy-2-methyl-3-oxo-3-(((S)-2-phenyl-1-(1H-tetrazol-5-yl)ethyl)amino)propyl)pyrrolidin-1-yl)-5-methyl-1-oxoheptan-4-yl)—N-methyl-2-((S)-3-methyl-2-(methylamino)butanamido)butanamide

To a stirred 23° C. suspension of Fmoc-MeVal-Abu(3-N₃)-Dil-OH TFA salt(0.491 g, 0.631 mmol) andH-Dap-(S)-2-Phenyl-1-(1H-tetrazol-5-yl)ethanamine TFA salt (0.532 g,1.13 mmol) in DMF (10 mL) was added DIEA (0.381 g, 0.500 mL, 2.97 mmol)followed by the addition of HATU (0.57 g, 1.486 mmol). After 10 h,analysis by LCMS showed the reaction was complete. The crude reactionwas diluted with saturated sodium bicarbonate (10 mL) and extracted withEtOAc (20 mL×3). The combined organic fractions were washed with brine,dried over a pad of magnesium sulfate, filtered, and concentrated invacuo. The crude oil was purified by preparatory RP-HPLC with aPhenomenex Gemini NX-C18 10μ 110 Å column (150×30 mm) using 10% to 90%MeCN in 0.1% aqueous formic acid as the eluent. A total of 125 mg ofFmoc-MeVal-Abu(3-N₃)-Dil-Dap-(S)-2-Phenyl-1-(1H-tetrazol-5-yl)ethanaminewas obtained as the formic acid salt (0.119 mmol, 16%). LCMS RT=1.94 min(Method B); ESI-MS m/z 1005.35 [M+H]⁺.

To a stirred 23° C. solution ofFmoc-MeVal-Abu(3-N₃)-Dil-Dap-(S)-2-Phenyl-1-(1H-tetrazol-5-yl)ethanamine (525 mg, 0.499 mmol) in acetonitrile (10 mL) was addedpiperidine (5 mL). After 2 h, analysis by LCMS showed the reaction wascomplete. To the crude reaction solution was added hexanes (×3). Theacetonitrile layer was concentrated in vacuo. The crude oil was purifiedby preparatory RP-HPLC with a Phenomenex Gemini NX-C18 10μ 110 Å column(150×30 mm) using 5% to 95% MeCN in 0.1% aqueous TFA as the eluent. Atotal of 20.0 mg of the title compound was obtained as the TFA salt(0.022 mmol, 5%). LCMS RT=1.37 min (Method B); ESI-MS m/z 783.42 [M+H]⁺;HRMS m/z 783.4979 [C₃₈H₆₂N₁₂O₆+H]⁺.

Example 61(2S,3S)-3-azido-2-((S)-2-(dimethylamino)-3-methylbutanamido)-N-((3R,4S,5S)-3-methoxy-1-((S)-2-((1R,2R)-1-methoxy-2-methyl-3-oxo-3-(((S)-2-phenyl-1-(thiazol-2-yl)ethyl)amino)propyl)pyrrolidin-1-yl)-5-methyl-1-oxoheptan-4-yl)-N-methylbutanamide

To a stirred 23° C. suspension of Boc-Dap-OH dicyclohexylamine salt(1.95 g, 4.154 mmol) and (S)-2-phenyl-1-(2-thiazol-2-yl)ethylamine (1.00g, 4.154 mmol) in CH₂Cl₁₂ (20.0 mL) was added DIEA (2.15 g, 2.9 mL,16.615 mmol) followed by the addition of DEPC (1.01 g, 0.9 mL, 0.006mol). After 10 h, analysis by LCMS showed the reaction was complete. Thecrude reaction mixture was washed with H₂O (25 mL×2), followed by brine(25 mL×2). The organic fraction was dried over a pad of MgSO₄, filteredand concentrated in vacuo. A total of 1.65 g ofBoc-Dap-(S)-2-phenyl-1-(thiazol-2-yl)ethanamine (3.48 mmol, 84%) wasobtained as a yellow oil. LCMS RT=1.59 min (Method B); ESI-MS m/z475.2[M+H]⁺.

To a stirred 23° C. suspension ofBoc-Dap-(S)-2-phenyl-1-(thiazol-2-yl)ethanamine (1.65 g, 3.49 mmol) inCH₂Cl₂ (10.0 mL) was added TFA (10.0 mL). After 4 h, analysis by LCMSshowed the reaction was complete. The volatile organics were evaporatedin vacuo. The crude oil was dissolved in DMF (5 mL) and triethylamine (1mL) to achieve a pH of 8. The crude oil was purified by preparatoryRP-HPLC with a Phenomenex Gemini NX-C18 10μ 110 Å column (150×30 mm)using 5% to 95% MeCN in 0.1% aqueous TFA as the eluent. A total of 935mg of H-Dap-(S)-2-phenyl-1-(thiazol-2-yl)ethanamine (1.92 mmol, 55%) wasobtained as the TFA salt. LCMS RT=1.04 min (Method B); ESI-MS m/z 375.0[M+H]⁺.

To a stirred 23° C. suspension of Dov-Abu(3-N₃)-Dil-OH TFA salt (302 mg,0.661 mmol) and H-Dap-(S)-2-phenyl-1-(thiazol-2-yl)ethanamine TFA salt(247 mg, 0.661 mmol) in DMF (10 mL) was added DIEA (0.34 g, 0.5 mL, 2.65mmol) followed by HATU (504 mg, 1.32 mmol). After 10 h, analysis by LCMSshowed the reaction was complete. The crude reaction mixture was dilutedwith saturated sodium bicarbonate (10 mL) and extracted with EtOAc (20mL×3). The combined organic fractions were washed with brine, dried overa pad of magnesium sulfate, filtered, and concentrated in vacuo. Thecrude oil was purified by preparatory RP-HPLC with a Phenomenex GeminiNX-C18 10μ 110 Å column (150×30 mm) using 10% to 90% MeCN in 0.1%aqueous TFA as the eluent. A total of 111 mg of the title compound wasobtained as the TFA salt (0.120 mmol, 18%). LCMS RT=1.33 min (Method B);ESI-MS m/z 812.2 [M+H]⁺; HRMS m/z 812.4835 [C₄₁H₆₅N₉O₆S+H]⁺.

Example 62 tert-butyl((2R,3R)-3-((S)-1-((3R,4S,5S)-4-((2S,3S)-3-amino-2-((S)-2-(dimethylamino)-3-methylbutanamido)-N-methylbutanamido)-3-methoxy-5-methylheptanoyl)pyrrolidin-2-yl)-3-methoxy-2-methylpropanoyl)-L-phenylalaninate

To a stirred room temperature solution ofDov-Abu(3-N₃)-Dil-Dap-Phe-Ot-Bu formic acid salt (102.5 mg, 0.117 mmol)in DMF (1.0 mL) was added trimethylphosphine in THF (1 M, 0.350 mL,0.350 mmol). After 2 h, analysis by LCMS showed that the reaction wascomplete. The crude reaction mixture was purified by preparatory RP-HPLCwith a Phenomenex Gemini-NX 10μ C-18 110 Å column (150×30 mm) using 5%to 95% MeCN in 0.1% aqueous formic acid as the eluent. A total of 70.1mg of the title compound was obtained as a formic acid salt (0.083 mmol,70%). LCMS RT=1.22 min (Method B); ESI-MS m/z 803.3 [M+H]⁺; HRMS m/z803.5642 [C₄₃H₇₄N₆O₈+H]⁺.

Example 63(2S,3S)-3-amino-2-((S)-2-(dimethylamino)-3-methylbutanamido)-N-((3R,4S,5S)-3-methoxy-1-((S)-2-((1R,2R)-1-methoxy-2-methyl-3-oxo-3-(((S)-2-phenyl-1-(1H-tetrazol-5-yl)ethyl)amino)propyl)pyrrolidin-1-yl)-5-methyl-1-oxoheptan-4-yl)-N-methylbutanamide

To a stirred room temperature solution ofDov-Abu(3-N₃)-Dil-Dap-Phe-Tetrazole formic acid salt (19.4 mg, 0.023mmol) in DMF (0.2 mL) was added trimethylphosphine in THF (1 M, 0.068mL, 0.068 mmol). After 2 h, analysis by LCMS showed that the reactionwas complete. The crude reaction mixture was purified by preparatoryRP-HPLC with a Phenomenex Gemini-NX 10μ C-18 110 Å column (150×30 mm)using 5% to 95% MeCN in 0.1% aqueous formic acid as the eluent. A totalof 9.9 mg of the title compound was obtained as a formic acid salt(0.012 mmol, 53%). LCMS RT=1.07 min (Method B); ESI-MS m/z 771.2 [M+H]⁺;HRMS m/z 771.5233 [C₃₉H₆₆N₁₀O₈+H]⁺.

Example 64(2S,3S)-3-amino-2-((S)-2-(dimethylamino)-3-methylbutanamido)-N-((3R,4S,5S)-3-methoxy-1-((S)-2-((1R,2R)-1-methoxy-2-methyl-3-oxo-3-(((S)-2-phenyl-1-(thiazol-2-yl)ethyl)amino)propyl)pyrrolidin-1-yl)-5-methyl-1-oxoheptan-4-yl)-N-methylbutanamide

To a stirred room temperature solution ofDov-Abu(3-N₃)-Dil-Dap-Phe-Thiazole formic acid salt (37.2 mg, 0.043mmol) in THF (0.2 mL) was added trimethylphosphine in THF (1 M, 0.090mL, 0.090 mmol). After 1 h, analysis by LCMS showed that the reactionwas complete. The crude reaction mixture was purified by preparatoryRP-HPLC with a Phenomenex Gemini-NX 10μ C-18 110 Å column (150×30 mm)using 5% to 95% MeCN in 0.1% aqueous formic acid as the eluent. A totalof 16.8 mg of the title compound was obtained as a formic acid salt(0.020 mmol, 47%). LCMS RT=1.15 min (Method B); ESI-MS m/z 786.2 [M+H]⁺;HRMS m/z 786.4940 [C₄₁H₆₇N₇O₆S+H]⁺.

Example B1 In Vitro Cytotoxicity Experiments

The in vitro efficacy of the compounds was measured by evaluating theircytotoxic activity on various cancer cell lines. This assay wasconducted in clear tissue-culture treated 96-well plates. The cell linesused were PC3 (human prostate carcinoma), HCC-1954 (human mammary ductalcarcinoma), and HCT15 (human colorectal adenocarcinoma, Pgp-expressing).Cells were seeded at approximately 1,000-1,500 cells per well in 50 μLof growth media (RPMI -1640+10% heat-inactivated fetal bovine serum) andincubated overnight at 37° C. with 5% CO₂ to allow them to attach. Thenext day, 50 μL of a 2× stock of vehicle control (DMSO) or compounds atvarying concentrations was added to each well in triplicate. Inaddition, control wells with no cells or untreated cells alone wereused. The plates were incubated in the humidified tissue cultureincubator with 5% CO₂ at 37° C. for 4 to 6 days after addition ofcompounds to measure cytotoxicity. After 4 to 6 days, 20 μL ofPrestoBlue™ Cell Viability Reagent (Life Technologies # A13261) wasadded per well. Plates were incubated at 37° C. for 1 to 2 h.Fluorescence was recorded at 540 ex/590 em using the Biotek Synergy™ H₄plate reader. Representative data is graphed as percent survivalcompared to untreated control wells. Data for compounds tested in thisassay are graphed as percent survival compared to untreated controlwells, as shown in FIGS. 9-30.

Example B2 Determination of Tubulin Polymerization

The inhibition of tubulin polymerization by the compounds describedherein was evaluated on bovine brain tubulin. To evaluate the activityof compounds, tubulin was seeded at approximately 400 g per well in 100μL of general tubulin buffer, and then treated with 10 M finalconcentration of compound in duplicate at the initiation of the assay.Tubulin polymerization assays were usually carried out at 37° C. for 60min after the addition of test compounds. Tubulin polymerization wasdetermined by absorbance spectroscopy using the optical density value at340 nm. To assess the amount of polymerized tubulin, the optical densityvalue at 340 nm was obtained each minute after the addition of testcompounds. For analysis, the extent of tubulin polymerization by thecompound-treated tubulin was compared to that of the control, which wasbuffer-treated tubulin. In particular, tubulin inhibition studies wereperformed using HTS-Tubulin Polymerization Assay Kit (Cytoskeleton Inc.;Catalog # BK004P), using the following sample protocol:

-   -   1. Pre-warm the spectrophotometer and 96-well plates to 37° C.        for 30 min prior to starting the assay. A warm plate is        essential for high polymerization activity and reproducible        results.    -   2. Enter all plate reader parameters (Absorbance at 340 nm, 37°        C., one read each minute) so that the spectrophotometer is ready        for use. Once the tubulin is aliquoted into the 37° C. wells,        the reading must begin immediately.    -   3. Warm 500 μL of general tubulin buffer to room temperature.        Warm buffer is needed for tubulin ligand dilutions.    -   4. Paclitaxel is included as a control. Use 10 μL of Paclitaxel        per well, which brings the final concentration to 10 μM final.    -   5. Make cold assay buffer: general tubulin buffer, 1 mM GTP, 10%        glycerol.    -   6. Resuspend 4 mgs of tubulin with 1 mL of cold assay buffer to        bring the final protein concentration of 4 mg/mL. Place the        tubes on ice and allow 3 min for the complete resuspension of        the protein.    -   7. Prepare selected compound at 10× concentration in assay        buffer.    -   8. Pipette 10 μL of the 10× concentrated compound into the        required number of wells of the pre-warmed plate. Incubate the        plate for 2 min at 37° C.    -   9. Pipette 10 μL of assay buffer only into two control wells        (buffer-treated tubulin).    -   10. Pipette 100 μL of tubulin into the required number of wells        (two wells should be the zero compound controls, which are        buffer-treated).    -   11. Immediately place the plate into the spectrophotometer at        37° C. and start recording the optical density at 340 nm each        minute. Increasing optical density values at 340 nm equate to        increasing tubulin polymerization.

Data for compounds tested in this assay are presented in FIGS. 1-8.

Example B3 Determination of In Vivo Efficacy of Test Compounds: EfficacyEvaluation in Subcutaneously Established Human Bladder Cancer Cell LineSW780 Implanted in ICR SCID Mice

For animal in vivo studies, the test compounds are diluted with 20 mMHistidine, 5% Sucrose, pH 6 with 15% DMSO. Male ICR SCID mice (TaconicFarm, Hudson, N.Y.) are housed in standard rodent micro isolator cages.Environment controls for the animal rooms are set to maintain atemperature between 20-24° C., a relative humidity between 30% to 70%,and an approximate 12 h light/12 h dark cycle. Food and water areprovided ad libitum. After 72 h of acclimatization, the mice areimplanted with SW780 human bladder cancer cells (2×10⁶ cells/mouse),suspended in 50% complete cultrex (Trevigen, Inc.) mixed with PBS(Gibco), and the tumor growth rate is monitored. When the average tumorvolume reaches ˜200 mm³, tumors are size-matched and mice are randomizedto treatment groups (n=8 or 10). The tumor-bearing mice are treated i.v.with Vehicle or test compound at 2 or 4 mg/kg (mpk) on a QW dosingschedule for 3 weeks. Tumor volume is assessed twice weekly usingcaliper measurement.

While the foregoing written description of the compounds, uses, andmethods described herein enables one of ordinary skill to make and usethe compounds, uses, and methods described herein, those of ordinaryskill will understand and appreciate the existence of variations,combinations, and equivalents of the specific embodiment, method, andexamples herein. The compounds, uses, and methods provided herein shouldtherefore not be limited by the above-described embodiments, methods, orexamples, but rather encompasses all embodiments and methods within thescope and spirit of the compounds, uses, and methods provided herein.

The invention claimed is:
 1. A compound of Formula (I):

wherein R¹ and R² are each independently —H or alkyl; X is absent; R³ isa group of the formula:

 wherein R¹⁵ and R¹⁶ are each independently —H, —OH, —NH₂, —SH, —N₃,alkyl, alkenyl, alkynyl, -alkyl-OH, -alkyl-NH₂, -alkyl-SH, or -alkyl-N₃;R⁴ is a group of the formula:

 wherein R¹⁷ is —OH, —NH₂, —SH, —N₃, —CO₂H, alkenyl, alkynyl, -alkyl-OH,-alkyl-NH₂, -alkyl-SH, -alkyl-N₃ or -alkyl-CO₂H; and R¹⁸ is —H, —OH,—NH₂, —SH, —N₃, —CO₂H, alkyl, alkenyl, alkynyl, -alkyl-OH, -alkyl-NH₂,-alkyl-SH, -alkyl-N₃ or -alkyl-CO₂H; R⁵ is sec-butyl or isobutyl; R⁶ is—H or alkyl; R⁷ is —H, alkyl, —CO₂R^(a), —CONR^(b)R^(c), substituted orunsubstituted phenyl, or substituted or unsubstituted heterocyclic ring;R⁸ is —H, alkyl, substituted or unsubstituted phenyl, or substituted orunsubstituted heterocyclic ring; wherein R^(a) is —H or alkyl; R^(b) andR^(c) are each independently —H or alkyl; R⁹ is —H or alkyl; or R⁹ istaken together with R⁴ and the atoms to which they are attached to forma substituted or unsubstituted heterocycloalkyl ring; R¹⁰ is —H oralkyl; R¹¹ is —H or alkyl; R¹² is —H or alkyl; R¹³ is —H or alkyl; andR¹⁴ is —H, —OH or alkyl; or a pharmaceutically acceptable salt thereof.2. The compound of claim 1, wherein R¹⁵ and R¹⁶ are each independently-Hor alkyl; R⁷ is —H, —CO₂R^(a), —CONR^(b)R^(c) or substituted orunsubstituted heterocyclic ring; R⁸ is substituted or unsubstitutedphenyl, or substituted or unsubstituted heterocyclic ring; and R¹⁴ is—H.
 3. The compound of claim 2, wherein R¹⁵ and R¹⁶ are each methyl. 4.The compound of claim 3, wherein R¹⁷ is —OH, —NH₂, —SH or —N₃; and R¹⁸is —H or alkyl.
 5. The compound of claim 4, wherein R⁷ is —H, —CO₂R^(a)or —CONR^(b)R^(c); and R⁸ is phenyl.
 6. The compound of claim 1, whereinthe compound is selected from the group consisting of: (S)-methyl2-((2R,3R)-3-((S)-1-((3R,4S,5S)-4-((S)-2-((S)-2-(dimethylamino)-3-methylbutanamido)-3-hydroxy-N-methylpropanamido)-3-methoxy-5-methylheptanoyl)pyrrolidin-2-yl)-3-methoxy-2-methylpropanamido)-3-phenylpropanoate;(S)-methyl2-((2R,3R)-3-((S)-1-((3R,4S,5S)-4-((2S,3R)-2-((S)-2-(dimethylamino)-3-methylbutanamido)-3-hydroxy-N-methylbutanamido)-3-methoxy-5-methylheptanoyl)pyrrolidin-2-yl)-3-methoxy-2-methylpropanamido)-3-phenylpropanoate;(S)-2-(dimethylamino)-N—((S)-3-hydroxy-1-(((3R,4S,5i)-3-methoxy-1-((S)-2-((1R,2R)-1-methoxy-2-methyl-3-oxo-3-((2-(pyridin-2-yl)ethyl)amino)propyl)pyrrolidin-1-yl)-5-methyl-1-oxoheptan-4-yl)(methyl)amino)-1-oxopropan-2-yl)-3-methylbutanamide;(2S,3R)-2-((S)-2-(dimethylamino)-3-methylbutanamido)-3-hydroxy-N-((3R,4S,5S)-3-methoxy-1-((S)-2-((1R,2R)-1-methoxy-2-methyl-3-oxo-3-((2-(pyridin-2-yl)ethyl)amino)propyl)pyrrolidin-1-yl)-5-methyl-1-oxoheptan-4-yl)-N-methylbutanamide;(2S)-2-(dimethylamino)-N-((2S)-3-hydroxy-1-(((3R,4S,5S)-3-methoxy-1-((2S)-2-((1R,2R)-1-methoxy-2-methyl-3-oxo-3-((2-(piperidin-2-yl)ethyl)amino)propyl)pyrrolidin-1-yl)-5-methyl-1-oxoheptan-4-yl)(methyl)amino)-1-oxopropan-2-yl)-3-methylbutanamide;(2S,3R)-2-((S)-2-(dimethylamino)-3-methylbutanamido)-3-hydroxy-N-((3R,4S,5S)-3-methoxy-1-((2S)-2-((1R,2R)-1-methoxy-2-methyl-3-oxo-3-((2-(piperidin-2-yl)ethyl)amino)propyl)pyrrolidin-1-yl)-5-methyl-1-oxoheptan-4-yl)-N-methylbutanamide;(S)-methyl 2-((2R,3R)-3-((S)-1-((3R,4S,5S)-4-((2S,3S)-3-azido-2-((S)-2-(dimethylamino)-3-methylbutanamido)-N-methylbutanamido)-3-methoxy-5-methylheptanoyl)pyrrolidin-2-yl)-3-methoxy-2-methylpropanamido)-3-phenylpropanoate;(S)-methyl2-((2R,3R)-3-((S)-1-((3R,4S,5S)-4-((S)-3-amino-2-((S)-2-(dimethylamino)-3-methylbutanamido)-N-methylpropanamido)-3-methoxy-5-methylheptanoyl)pyrrolidin-2-yl)-3-methoxy-2-methylpropanamido)-3-phenylpropanoate;(S)—N—((S)-3-amino-1-(((3R,4S,5S)-3-methoxy-1-((S)-2-((1R,2R)-1-methoxy-2-methyl-3-oxo-3-((2-(pyridin-2-yl)ethyl)amino)propyl)pyrrolidin-1-yl)-5-methyl-1-oxoheptan-4-yl)(methyl)amino)-1-oxopropan-2-yl)-2-(dimethylamino)-3-methylbutanamide;(S)-methyl2-((2R,3R)-3-((S)-1-((3R,4S,5S)-4-((S)-3-azido-2-((S)-2-(dimethylamino)-3-methylbutanamido)-N-methylpropanamido)-3-methoxy-5-methylheptanoyl)pyrrolidin-2-yl)-3-methoxy-2-methylpropanamido)-3-phenylpropanoate;(S)-methyl2-((2R,3R)-3-((S)-1-((3R,4S,5S)-4-((S)-4-azido-2-((S)-2-(dimethylamino)-3-methylbutanamido)-N-methylbutanamido)-3-methoxy-5-methylheptanoyl)pyrrolidin-2-yl)-3-methoxy-2-methylpropanamido)-3-phenylpropanoate;(S)-methyl2-((2R,3R)-3-((S)-1-((3R,4S,5S)-4-((S)-4-amino-2-((S)-2-(dimethylamino)-3-methylbutanamido)-N-methylbutanamido)-3-methoxy-5-methylheptanoyl)pyrrolidin-2-yl)-3-methoxy-2-methylpropanamido)-3-phenylpropanoate;(S)-2-((2R,3R)-3-((S)-1-((3R,4S,5S)-4-((2S,3S)-3-azido-2-((S)-2-(dimethylamino)-3-methylbutanamido)-N-methylbutanamido)-3-methoxy-5-methylheptanoyl)pyrrolidin-2-yl)-3-methoxy-2-methylpropanamido)-3-phenylpropanoic acid; (S)-methyl2-((2R,3R)-3-((S)-1-((3R,4S,5S)-4-((R)-2-((S)-2-(dimethylamino)-3-methylbutanamido)-3-mercapto-N-methylpropanamido)-3-methoxy-5-methylheptanoyl)pyrrolidin-2-yl)-3-methoxy-2-methylpropanamido)-3-phenylpropanoate;(S)-2-((2R,3R)-3-((S)-1-((3R,4S,5S)-4-((R)-2-((S)-2-(dimethylamino)-3-methylbutanamido)-3-mercapto-N-methylpropanamido)-3-methoxy-5-methylheptanoyl)pyrrolidin-2-yl)-3-methoxy-2-methylpropanamido)-3-phenylpropanoicacid;(S)-2-((2R,3R)-3-((S)-1-((3R,4S,5S)-4-((S)-2-((S)-2-(dimethylamino)-3-methylbutanamido)-3-hydroxy-N-methylpropanamido)-3-methoxy-5-methylheptanoyl)pyrrolidin-2-yl)-3-methoxy-2-methylpropanamido)-3-phenylpropanoicacid;(S)-2-((2R,3R)-3-((S)-1-((3R,4S,5S)-4-((2S,3R)-2-((S)-2-(dimethylamino)-3-methylbutanamido)-3-hydroxy-N-methylbutanamido)-3-methoxy-5-methylheptanoyl)pyrrolidin-2-yl)-3-methoxy-2-methylpropanamido)-3-phenylpropanoicacid; (S)-methyl2-((2R,3R)-3-((S)-1-((3R,4S,5S)-4-((2S,3S)-3-amino-2-((S)-2-(dimethylamino)-3-methylbutanamido)-N-methylbutanamido)-3-methoxy-5-methylheptanoyl)pyrrolidin-2-yl)-3-methoxy-2-methylpropanamido)-3-phenylpropanoate;(S)-2-((2R,3R)-3-((S)-1-((3R,4S,5S)-4-((2S,3S)-3-amino-2-((S)-2-(dimethylamino)-3-methylbutanamido)-N-methylbutanamido)-3-methoxy-5-methylheptanoyl)pyrrolidin-2-yl)-3-methoxy-2-methylpropanamido)-3-phenylpropanoic acid;(2S,3S)-3-azido-2-((S)-2-(dimethylamino)-3-methylbutanamido)-N-((3R,4S,5S)-3-methoxy-1-((S)-2-((1R,2R)-1-methoxy-2-methyl-3-oxo-3-(phenethylamino)propyl)pyrrolidin-1-yl)-5-methyl-1-oxoheptan-4-yl)-N-methylbutanamide;(2S,3S)-3-azido-2-((S)-2-(dimethylamino)-3-methylbutanamido)-N-((3R,4S,5S)-1-((S)-2-((1R,2R)-3-(((1S,2R)-1-hydroxy-1-phenylpropan-2-yl)amino)-1-methoxy-2-methyl-3-oxopropyl)pyrrolidin-1-yl)-3-methoxy-5-methyl-1-oxoheptan-4-yl)-N-methylbutanamide;(2S,3S)-3-azido-N-((3R,4S,5S)-1-((S)-2-((1R,2R)-3-((4-chlorophenethyl)amino)-1-methoxy-2-methyl-3-oxopropyl)pyrrolidin-1-yl)-3-methoxy-5-methyl-1-oxoheptan-4-yl)-2-((S)-2-(dimethylamino)-3-methylbutanamido)-N-methylbutanamide;(2S,3S)-3-azido-N-((3R,4S,5S)-1-((S)-2-((1R,2R)-3-((2-chlorophenethyl)amino)-1-methoxy-2-methyl-3-oxopropyl)pyrrolidin-1-yl)-3-methoxy-5-methyl-1-oxoheptan-4-yl)-2-((S)-2-(dimethylamino)-3-methylbutanamido)-N-methylbutanamide;(2S,3S)-3-amino-2-((S)-2-(dimethylamino)-3-methylbutanamido)-N-((3R,4S,5S)-3-methoxy-1-((S)-2-((1R,2R)-1-methoxy-2-methyl-3-oxo-3-(phenethylamino)propyl)pyrrolidin-1-yl)-5-methyl-1-oxoheptan-4-yl)-N-methylbutanamide;(2S,3S)-3-amino-2-((S)-2-(dimethylamino)-3-methylbutanamido)-N-((3R,4S,5S)-1-((S)-2-((1R,2R)-3-(((1S,2R)-1-hydroxy-1-phenylpropan-2-yl)amino)-1-methoxy-2-methyl-3-oxopropyl)pyrrolidin-1-yl)-3-methoxy-5-methyl-1-oxoheptan-4-yl)-N-methylbutanamide;(2S,3S)-3-amino-N-((3R,4S,5S)-1-((S)-2-((1R,2R)-3-((4-chlorophenethyl)amino)-1-methoxy-2-methyl-3-oxopropyl)pyrrolidin-1-yl)-3-methoxy-5-methyl-1-oxoheptan-4-yl)-2-((S)-2-(dimethylamino)-3-methylbutanamido)-N-methylbutanamide;(2S,3S)-3-amino-N-((3R,4S,5S)-1-((S)-2-((1R,2R)-3-((2-chlorophenethyl)amino)-1-methoxy-2-methyl-3-oxopropyl)pyrrolidin-1-yl)-3-methoxy-5-methyl-1-oxoheptan-4-yl)-2-((S)-2-(dimethylamino)-3-methylbutanamido)-N-methylbutanamide;(S)-4-amino-2-((S)-2-(dimethylamino)-3-methylbutanamido)-N-((3R,4S,5S)-3-methoxy-1-((S)-2-((1R,2R)-1-methoxy-2-methyl-3-oxo-3-(phenethylamino)propyl)pyrrolidin-1-yl)-5-methyl-1-oxoheptan-4-yl)-N-methylbutanamide;(S)-4-amino-2-((S)-2-(dimethylamino)-3-methylbutanamido)-N-((3R,4S,5S)-1-((S)-2-((1R,2R)-3-(((1S,2R)-1-hydroxy-1-phenylpropan-2-yl)amino)-1-methoxy-2-methyl-3-oxopropyl)pyrrolidin-1-yl)-3-methoxy-5-methyl-1-oxoheptan-4-yl)-N-methylbutanamide;(S)-4-amino-N-((3R,4S,5S)-1-((S)-2-((1R,2R)-3-((4-chlorophenethyl)amino)-1-methoxy-2-methyl-3-oxopropyl)pyrrolidin-1-yl)-3-methoxy-5-methyl-1-oxoheptan-4-yl)-2-((S)-2-(dimethylamino)-3-methylbutanamido)-N-methylbutanamide;(S)-4-amino-N-((3R,4S,5S)-1-((S)-2-((1R,2R)-3-((2-chlorophenethyl)amino)-1-methoxy-2-methyl-3-oxopropyl)pyrrolidin-1-yl)-3-methoxy-5-methyl-1-oxoheptan-4-yl)-2-((S)-2-(dimethylamino)-3-methylbutanamido)-N-methylbutanamide;methyl((2R,3R)-3-((S)-1-((3R,4S,5S)-4-((S)-2-((S)-2-(dimethylamino)-3-methylbutanamido)-N-methylpent-4-ynamido)-3-methoxy-5-methylheptanoyl)pyrrolidin-2-yl)-3-methoxy-2-methylpropanoyl)-L-phenylalaninate;(2S,3S)—N-((3R,4S,5S)-1-((S)-2-((1R,2R)-3-(((S)-1-amino-1-oxo-3-phenylpropan-2-yl)amino)-1-methoxy-2-methyl-3-oxopropyl)pyrrolidin-1-yl)-3-methoxy-5-methyl-1-oxoheptan-4-yl)-3-azido-2-((S)-2-(dimethylamino)-3-methylbutanamido)-N-methylbutanamide;(2S,3S)-3-azido-2-((S)-2-(dimethylamino)-3-methylbutanamido)-N-((3R,4S,5S)-3-methoxy-1-((S)-2-((1R,2R)-1-methoxy-2-methyl-3-oxo-3-((2-(pyridin-2-yl)ethyl)amino)propyl)pyrrolidin-1-yl)-5-methyl-1-oxoheptan-4-yl)-N-methylbutanamide;(2S,3S)-3-azido-N-((3R,4S,5S)-1-((S)-2-((1R,2R)-3-(((S)-1-(tert-butylamino)-1-oxo-3-phenylpropan-2-yl)amino)-1-methoxy-2-methyl-3-oxopropyl)pyrrolidin-1-yl)-3-methoxy-5-methyl-1-oxoheptan-4-yl)-2-((S)-2-(dimethylamino)-3-methylbutanamido)-N-methylbutanamide;methyl((2R,3R)-3-((S)-1-((3R,4S,5S)-4-((2S,3S)-3-azido-2-((S)-2-(dimethylamino)-3-methylbutanamido)-N-methylbutanamido)-3-methoxy-5-methylheptanoyl)pyrrolidin-2-yl)-3-methoxy-2-methylpropanoyl)-L-valinate; methyl((2R,3R)-3-((S)-1-((3R,4S,5S)-4-((S)-6-amino-2-((S)-2-(dimethylamino)-3-methylbutanamido)-N-methylhexanamido)-3-methoxy-5-methylheptanoyl)pyrrolidin-2-yl)-3-methoxy-2-methylpropanoyl)-L-phenylalaninate;(S)-3-((S)-2-(dimethylamino)-3-methylbutanamido)-4-(((3R,4S,5S)-3-methoxy-1-((S)-2-((1R,2R)-1-methoxy-3-(((S)-1-methoxy-1-oxo-3-phenylpropan-2-yl)amino)-2-methyl-3-oxopropyl)pyrrolidin-1-yl)-5-methyl-1-oxoheptan-4-yl)(methyl)amino)-4-oxobutanoicacid;(2S,3R)-2-((S)-2-(dimethylamino)-3-methylbutanamido)-3-hydroxy-N-((3R,4S,5S)-1-((S)-2-((1R,2R)-3-(((1S,2R)-1-hydroxy-1-phenylpropan-2-yl)amino)-1-methoxy-2-methyl-3-oxopropyl)pyrrolidin-1-yl)-3-methoxy-5-methyl-1-oxoheptan-4-yl)-N-methylbutanamide;methyl((2R,3R)-3-((S)-1-((3R,4S,5S)-4-((2S,3S)-3-azido-2-((S)-2-(dimethylamino)-3-methylbutanamido)-N-methylbutanamido)-3-methoxy-5-methylheptanoyl)pyrrolidin-2-yl)-3-methoxy-2-methylpropanoyl)-L-isoleucinate;(2S,3S)-3-amino-N-((3R,4S,5S)-1-((S)-2-((1R,2R)-3-(((S)-1-amino-1-oxo-3-phenylpropan-2-yl)amino)-1-methoxy-2-methyl-3-oxopropyl)pyrrolidin-1-yl)-3-methoxy-5-methyl-1-oxoheptan-4-yl)-2-((S)-2-(dimethylamino)-3-methylbutanamido)-N-methylbutanamide;(2S,3S)-3-amino-N-((3R,4S,5S)-1-((S)-2-((1R,2R)-3-(((S)-1-(tert-butylamino)-1-oxo-3-phenylpropan-2-yl)amino)-1-methoxy-2-methyl-3-oxopropyl)pyrrolidin-1-yl)-3-methoxy-5-methyl-1-oxoheptan-4-yl)-2-((S)-2-(dimethylamino)-3-methylbutanamido)-N-methylbutanamide;methyl((2R,3R)-3-((S)-1-((3R,4S,5S)-4-((S)-3-azido-N-methyl-2-((S)-3-methyl-2-(methylamino)butanamido)propanamido)-3-methoxy-5-methylheptanoyl)pyrrolidin-2-yl)-3-methoxy-2-methylpropanoyl)-L-phenylalaninate;methyl((2R,3R)-3-((S)-1-((3R,4S,5S)-4-((2S,3S)-3-azido-N-methyl-2-((S)-3-methyl-2-(methylamino)butanamido)butanamido)-3-methoxy-5-methylheptanoyl)pyrrolidin-2-yl)-3-methoxy-2-methylpropanoyl)-L-phenylalaninate;(2S,3S)—N-((3R,4S,5S)-1-((S)-2-((1R,2R)-3-(((S)-1-amino-1-oxo-3-phenylpropan-2-yl)amino)-1-methoxy-2-methyl-3-oxopropyl)pyrrolidin-1-yl)-3-methoxy-5-methyl-1-oxoheptan-4-yl)-3-azido-N-methyl-2-((S)-3-methyl-2-(methylamino)butanamido)butanamide;((2S,3S)-3-azido-N-((3R,4S,5S)-1-((S)-2-((1R,2R)-3-(((S)-1-(tert-butylamino)-1-oxo-3-phenylpropan-2-yl)amino)-1-methoxy-2-methyl-3-oxopropyl)pyrrolidin-1-yl)-3-methoxy-5-methyl-1-oxoheptan-4-yl)-N-methyl-2-((S)-3-methyl-2-(methylamino)butanamido)butanamide;tert-butyl((2R,3R)-3-((S)-1-((3R,4S,5S)-4-((2S,3S)-3-azido-N-methyl-2-((S)-3-methyl-2-(methylamino)butanamido)butanamido)-3-methoxy-5-methylheptanoyl)pyrrolidin-2-yl)-3-methoxy-2-methylpropanoyl)-L-phenylalaninate;((2R,3R)-3-((S)-1-((3R,4S,5S)-4-((2S,3S)-3-azido-N-methyl-2-((S)-3-methyl-2-(methylamino)butanamido)butanamido)-3-methoxy-5-methylheptanoyl)pyrrolidin-2-yl)-3-methoxy-2-methylpropanoyl)-L-phenylalanine;tert-butyl((2R,3R)-3-((S)-1-((3R,4S,5S)-4-((2S,3S)-3-azido-2-((S)-2-(dimethylamino)-3-methylbutanamido)-N-methylbutanamido)-3-methoxy-5-methylheptanoyl)pyrrolidin-2-yl)-3-methoxy-2-methylpropanoyl)-L-phenylalaninate;(2S,3S)-3-azido-2-((S)-2-(dimethylamino)-3-methylbutanamido)-N-((3R,4S,5S)-3-methoxy-1-((S)-2-((1R,2R)-1-methoxy-2-methyl-3-oxo-3-(((S)-2-phenyl-1-(1H-tetrazol-5-yl)ethyl)amino)propyl)pyrrolidin-1-yl)-5-methyl-1-oxoheptan-4-yl)-N-methylbutanamide;(2S,3S)-3-azido-N-((3R,4S,5S)-3-methoxy-1-((S)-2-((1R,2R)-1-methoxy-2-methyl-3-oxo-3-(((S)-2-phenyl-1-(1H-tetrazol-5-yl)ethyl)amino)propyl)pyrrolidin-1-yl)-5-methyl-1-oxoheptan-4-yl)-N-methyl-2-((S)-3-methyl-2-(methylamino)butanamido)butanamide;(2S,3S)-3-azido-2-((S)-2-(dimethylamino)-3-methylbutanamido)-N-((3R,4S,5S)-3-methoxy-1-((S)-2-((1R,2R)-1-methoxy-2-methyl-3-oxo-3-(((S)-2-phenyl-1-(thiazol-2-yl)ethyl)amino)propyl)pyrrolidin-1-yl)-5-methyl-1-oxoheptan-4-yl)-N-methylbutanamide;tert-butyl((2R,3R)-3-((S)-1-((3R,4S,5S)-4-((2S,3S)-3-amino-2-((S)-2-(dimethylamino)-3-methylbutanamido)-N-methylbutanamido)-3-methoxy-5-methylheptanoyl)pyrrolidin-2-yl)-3-methoxy-2-methylpropanoyl)-L-phenylalaninate;(2S,3S)-3-amino-2-((S)-2-(dimethylamino)-3-methylbutanamido)-N-((3R,4S,5S)-3-methoxy-1-((S)-2-((1R,2R)-1-methoxy-2-methyl-3-oxo-3-(((S)-2-phenyl-1-(1H-tetrazol-5-yl)ethyl)amino)propyl)pyrrolidin-1-yl)-5-methyl-1-oxoheptan-4-yl)-N-methylbutanamide;and(2S,3S)-3-amino-2-((S)-2-(dimethylamino)-3-methylbutanamido)-N-((3R,4S,5S)-3-methoxy-1-((S)-2-((1R,2R)-1-methoxy-2-methyl-3-oxo-3-(((S)-2-phenyl-1-(thiazol-2-yl)ethyl)amino)propyl)pyrrolidin-1-yl)-5-methyl-1-oxoheptan-4-yl)-N-methylbutanamide;or a pharmaceutically acceptable salt thereof.
 7. The compound orpharmaceutically acceptable salt of claim 6, wherein the compound isselected from the group consisting of:(S)-2-((S)-2-(aminooxy)-3-methylbutanamido)-N-((3R,4S,5S)-3-methoxy-1-((S)-2-((1R,2R)-1-methoxy-2-methyl-3-oxo-3-((2-(pyridin-2-yl)ethyl)amino)propyl)pyrrolidin-1-yl)-5-methyl-1-oxoheptan-4-yl)-N,3-dimethylbutanamide;((2R,3R)-3-((S)-1-((3R,4S,5S)-4-((S)-2-((S)-2-(dimethylamino)-3-hydroxypropanamido)-N,3-dimethylbutanamido)-3-methoxy-5-methylheptanoyl)pyrrolidin-2-yl)-3-methoxy-2-methylpropanoyl)-L-phenylalanine;((2R,3R)-3-((S)-1-((3R,4S,5S)-4-((S)-2-((2S,3R)-2-(dimethylamino)-3-hydroxybutanamido)-N,3-dimethylbutanamido)-3-methoxy-5-methylheptanoyl)pyrrolidin-2-yl)-3-methoxy-2-methylpropanoyl)-L-phenylalanine;(S)-methyl2-((2R,3R)-3-((S)-1-((3R,4S,5S)-4-((S)-2-((S)-2-(dimethylamino)-3-hydroxypropanamido)-N,3-dimethylbutanamido)-3-methoxy-5-methylheptanoyl)pyrrolidin-2-yl)-3-methoxy-2-methylpropanamido)-3-phenylpropanoate;and (S)-methyl2-((2R,3R)-3-((S)-1-((3R,4S,5S)-4-((S)-2-((2S,3R)-2-(dimethylamino)-3-hydroxybutanamido)-N,3-dimethylbutanamido)-3-methoxy-5-methylheptanoyl)pyrrolidin-2-yl)-3-methoxy-2-methylpropanamido)-3-phenylpropanoate.
 8. A pharmaceuticalcomposition comprising an effective amount of a compound orpharmaceutically acceptable salt of claim 1 and a pharmaceuticallyacceptable excipient.
 9. A method of treating cancer, comprisingadministering to a subject in need thereof an effective amount of thecompound or pharmaceutically acceptable salt of claim
 1. 10. Thecompound or pharmaceutically acceptable salt of claim 1, wherein R¹ andR² are each independently —H or C₁₋₆-alkyl; R³ is

wherein R¹⁵ and R¹⁶ are each independently —H, —OH, or C₁₋₆-alkyl; R⁴ is

wherein R¹⁷ is —OH, —NH₂, —SH, —N₃, —CO₂H, C₁₋₆-alkyl-NH₂, alkynyl,alkenyl, or —C₁₋₆-alkyl-N₃; and R¹⁸ is —H or C₁₋₆-alkyl; R⁵ issec-butyl; R⁶ is —H; R⁷ is —H, C₁₋₆-alkyl, —CO₂R^(a), —CONR^(b)R¹⁰,tetrazolyl or thiazolyl; wherein R^(a) is —H or C₁₋₆-alkyl; and R^(b)and R^(c) are each independently —H or C₁₋₆-alkyl; R⁸ is —H, C₁₋₆-alkyl,substituted or unsubstituted phenyl or substituted or unsubstitutedheterocyclic ring; R⁹ is —H; R¹⁰, R¹¹, R¹², and R¹³ are eachindependently C₁₋₆-alkyl; and R¹⁴ is —H, C₁₋₆-alkyl or —OH.
 11. Thecompound or pharmaceutically acceptable salt of claim 1, wherein R¹ andR² are each independently —H or methyl; R³ is

wherein R¹⁵ and R¹⁶ are each independently —H, —OH, or methyl; R⁴ is

wherein R¹⁷ is —OH, —NH₂, —SH, —N₃, —CO₂H, aminomethyl, alkynyl,alkenyl, or azidomethyl; and R¹⁸ is —H or methyl; R⁵ is sec-butyl; R⁶ is—H; R⁷ is —H, methyl, —CO₂R^(a), or —CONR^(b)R^(c); wherein R^(a) is —Hor methyl; and R^(b) and R^(c) are each independently —H or methyl; R⁸is —H, methyl, ethyl, pyridinyl, piperidinyl, unsubstituted phenyl,phenyl substituted with halo; R⁹ is —H; R¹⁰, R¹¹, R¹², and R¹³ are eachmethyl; and R¹⁴ is —H, methyl or —OH.
 12. The compound orpharmaceutically acceptable salt of claim 1, wherein R¹ and R² are eachindependently —H or C₁₋₆-alkyl; R³ is

wherein R¹⁵ and R¹⁶ are each independently —H, —OH, or C₁₋₆-alkyl; R⁴ is

wherein R¹⁷ is —N₃; and R¹⁸ is —H or methyl; R⁵ is sec-butyl; R⁶ is —H;R⁷ is —H, C₁₋₆-alkyl, —CO₂R^(a), —CONR^(b)R^(c), tetrazolyl orthiazolyl; wherein R^(a) is —H or C₁₋₆-alkyl; and R^(b) and R^(c) areeach independently —H or C₁₋₆-alkyl; R⁸ is —H, C₁₋₆-alkyl, substitutedor unsubstituted phenyl or substituted or unsubstituted heterocyclicring; R⁹ is —H; R¹⁰, R¹¹, R¹², and R¹³ are each independentlyC₁₋₆-alkyl; and R¹⁴ is —H, C₁₋₆-alkyl or —OH.
 13. The compound orpharmaceutically acceptable salt of claim 1, wherein R¹ and R² are eachmethyl; R³ is a group of the formula:

wherein R¹⁵ and R¹⁶ are each methyl; R⁴ is a group of formula:

wherein R¹⁷ is —N₃, —NH₂, —OH, —SH, and R¹⁸ is —H or methyl; R⁵ issec-butyl; R⁶ is —H; R⁷ is —CO₂R^(a), or —CONR^(b)R^(c), wherein R^(a)is —H or C₁₋₆-alkyl; R^(b) and R^(c) are each independently —H orC₁₋₆-alkyl; R⁸ is phenyl; R⁹ is —H; R¹⁰, R¹¹, R¹², and R¹³ are eachmethyl; and R¹⁴ is —H.
 14. The compound or pharmaceutically acceptablesalt of claim 1, wherein R¹ and R² are each independently —H orC₁₋₆-alkyl; R³ is

wherein R¹⁵ and R¹⁶ are each independently —H, —OH, or C₁₋₆-alkyl; R⁴ is

wherein R¹⁷ is —N₃; and R¹⁸ is —H or C₁₋₆-alkyl; R⁵ is sec-butyl; R⁶ is—H; R⁷ is C₁₋₆-alkyl, —CONR^(b)R^(c), tetrazolyl or thiazolyl; whereinR^(b) and R^(c) are each independently —H or C₁₋₆-alkyl; R⁸ is —H,C₁₋₆-alkyl, substituted or unsubstituted phenyl or substituted orunsubstituted heterocyclic ring; R⁹ is —H; R¹⁰, R¹¹, R¹², and R¹³ areeach independently C₁₋₆-alkyl; and R¹⁴ is —H, C₁₋₆-alkyl or —OH.
 15. Thecompound or pharmaceutically acceptable salt of claim 1, wherein R¹ andR² are each independently —H or C₁₋₆-alkyl; R³ is

wherein R¹⁵ and R¹⁶ are each independently —H, —OH, or C₁₋₆-alkyl; R⁴ is

wherein R¹⁷ is —N₃; and R¹¹ is —H or C₁₋₆-alkyl; R⁵ is sec-butyl; R⁶ is—H; R⁷ is —CONR^(b)R^(c), wherein R^(b) and R^(c) are each independently—H or C₁₋₆-alkyl; R⁸ is —H, C₁₋₆-alkyl, substituted or unsubstitutedphenyl or substituted or unsubstituted heterocyclic ring; R⁹ is —H; R¹⁰,R¹¹, R¹², and R¹³ are each independently C₁₋₆-alkyl; and R¹⁴ is —H,C₁₋₆-alkyl or —OH.
 16. The compound or pharmaceutically acceptable saltof claim 1, wherein the compound is(2S,3S)—N-((3R,4S,5S)-1-((S)-2-((1R,2R)-3-(((S)-1-amino-1-oxo-3-phenylpropan-2-yl)amino)-1-methoxy-2-methyl-3-oxopropyl)pyrrolidin-1-yl)-3-methoxy-5-methyl-1-oxoheptan-4-yl)-3-azido-N-methyl-2-((S)-3-methyl-2-(methylamino)butanamido)butanamide.